Supplemental text file, including:

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1 Supplemental text file, including: Supplemental Table S1: Kinase profile for XL147 Supplemental Table S2: Effects of XL147 on PI3K Pathway Signaling in MCF7 and PC-3 Cells Supplemental Table S3: XL147 potency on cell proliferation by genotype Supplemental Table S4: Effects of XL147 on Cell Cycle of MCF7 Cells Supplemental Table S5: XL147 plasma concentrations, pharmacodynamics assays Supplemental Table S6: Efficacy of XL147 in U-87 MG, A549, A2058, and WM xenograft tumors Supplemental Figure Legends (Figures S1 to S6) MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 1

2 Supplemental Table S1 Kinase profile for XL147 a Kinase Axl TrkA IC 50 (nm) a a Abl; Akt1; Akt2; ALK; ALK4; Arg; ASK1; Aurora-A; Blk; B-Raf; BRK; Btk; CamK2 ; CaMKIV; CDK1/cyclinB; CDK2/cyclinA; CDK2/cyclinE; CDK3/cyclinE; CDK5/p35; CDK6/cyclinD3; CDK7/cyclinH/MAT; CHK1; CHK2; ChoK; CK1 ; CK2; c-raf; CSK; DDR2; EGFR; EMK; EphA2; EphA4; EphB4; ErbB2; ErbB4; FAK; Fer; Fes; FGFR1; FGFR2; FGFR3; FGFR4; Fgr; Flt-1; Flt-3; Flt-4; Fyn; GRK2; GSK3α; GSK3β; Hck; HIPK2; hpka; IGF-1R; IKKα; IKKβ; IRAK4; IRK; JAK2; JNK1α1; JNK2α2; JNK3; KDR; Kit; Lck; Lyn; MAP3K8; >10000 b MAP4K3; MAPK1; MAPK2; MAPKAP-K3; Met; MINK; MKK4; MKK6; MKK7β; MST1; MuSK; NEK2; NEK7; p38α; p38β; p38γ ; p70s6k; PAK1; PAK2; PAK4; PAK6; PDGFRβ; PDGFRα; PDK1; PIM1; PIM2; PIM3; PKCα; PKCβI; PKCβII; PKCγ; PKC ; PKC ; PKCµ; PKD2; Plk1; Plk3; PRAK; Pyk2; Ret; RIPK2; ROCK-I, ROCK-II; RON; Ros; Rse; SGK2; SphK; SYK; SRC;TAK1; TBK1; Tie-2; TrkA; TrkB; TSSK1; Yes; ZAP-70; ZIPK a Assays were performed at Exelixis or by Upstate (Charlottesville, VA) and the experimental methods are on file. b Highest concentration tested. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 2

3 Supplemental Table S2 Effects of XL147 on PI3K Pathway Signaling in MCF7 and PC-3 Cells a Protein (phospho-epitope) MCF7 (IC 50 nm) PC-3 (IC 50 nm) 0% Serum+EGF 10% Serum 0% Serum+EGF 10% Serum pakt T pakt S pp70s6k T % inhibition at 1200 nm 6790 ps6 S240/S % b % b ppras40 T pgsk3β S p4ebp1 T37/T46 74% b 43% b 23% b 13% b Cyclin D1 49% b 0% b 33% b 14% b perk Y204 58% b 27% b 0% b 0% b a See Materials and Methods for assay details. b Inhibition at nm. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 3

4 Supplemental Table S3 XL147 potency on cell proliferation by genotype XL147 XL147 Cell Line IC50 (nm) IC 50 (Relative) Genotype (PIK3CA, KRAS2, NRAS, BRAF, PTEN) BT PIK3CA mutation K111N (atypical may not be activating) T47D PIK3CA activating mutation H1047R IGROV PIK3A *1069_*1069insWKDN* (atypical) / PTEN mutation OVCAR PIK3CA gene amplification MCF PIK3CA activating mutation E545K MDA-MB PIK3CA activating mutation H1047R / PTEN mutation H PIK3CA activating mutation E545K / BRAF mutation G596R SK-OV PIK3CA activating mutation H1047R AGS PIK3CA activating mutation E453K / KRAS2 mutation G12D LS174T PIK3CA activating mutation H1047R / KRAS2 mutation G12D H460 > PIK3CA activating mutation E545K / KRAS2 mutation Q61H HCT116 > PIK3CA activating mutation H1047R / KRAS2 mutation G13D ZR PTEN mutation JRT3-T PTEN mutation PC PTEN mutation LNCaP PTEN mutation U87-MG PTEN mutation H PTEN mutation U PTEN mutation 786-O > PTEN mutation BC-3 > PTEN mutation H4 > PTEN mutation MDA-MB-468 > PTEN mutation OPM-2 > PTEN mutation U251 > PTEN mutation U373 > PTEN mutation MOLT NRAS mutation G12C / PTEN mutation WM BRAF mutation V600D / PTEN mutation A2058 > BRAF mutation V600E / PTEN mutation SK-MEL-28 > BRAF mutation V600E / PTEN mutation SK-MEL BRAF mutation V600E A375 > BRAF mutation V600E Colo-205 > BRAF mutation V600E HT29 > BRAF mutation V600E SK-HEP-1 > BRAF mutation V600E HL NRAS mutation Q61L OCI-AML NRAS mutation Q61L OVCAR KRAS2 mutation G12V RPMI KRAS2 mutation G12A A KRAS2 mutation G12S LoVo KRAS2 mutation G13D PANC KRAS2 mutation G12D Capan KRAS2 mutation G12V HT-1080 > NRAS mutation Q61K TALL-1 > NRAS mutation G12D CALU-6 > KRAS2 mutation Q61K MDA-MB-231T > KRAS2 mutation G13D / BRAF G464V H441 > KRAS2 mutation G12V SHP-77 > KRAS2 mutation G12V SW480 > KRAS2 mutation G12V AsPC-1 > KRAS2 mutation G12D DLD-1 > KRAS2 mutation G13D MiaPaCa > KRAS2 mutation G12C CESS MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 4

5 SNU NCI-N SK-BR KATO III H SNU KMS Caki H TF RL UACC SR Hs746T PL BXPC OE KG TT HPB-ALL Daoy SKM HN SK-N-SH SNU MOLT A Hep G A431 > DU145 > H69 > ALL-SIL > ARH-77 > Caco-2 > Caki CRO-AP5 > EHEB > GDM-1 > H187 > H345 > H510A > H526 > H82 > HeLa > HT > HuNS1 > K562 > MCF10A > MKN-45 > MNNG > Namalwa > OCI-AML5 > SW48 > Toledo > Note: Cytotoxicity IC 50 (nm) and Apoptosis EC 50 (nm) > nm for PC-3, MCF7, A549, LS174T, MDA-MB-468, U87-MG, and OVCAR-3 cells. Cell line genotype status are from Exelixis internal sequencing supplemented with data from COSMIC database. IGROV-1 PIK3CA status is from Whyte and Holbeck, Biochem Biophys Res Commun. 340: (2006). MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 5

6 Supplemental Table S4 Effects of XL147 on Cell Cycle of MCF7 Cells a 48 h [XL147] (µm) % Sub-G1 % G1 % S % G2/M h [XL147] (µm) % Sub-G1 % G1 % S % G2/M a See Materials and Methods for assays details. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 6

7 Supplemental Table S5 XL147 plasma concentrations, pharmacodynamics assays Time-point (h) 4 24 Dose (mg/kg) Plasma Concentration Mean SD (μm) MCF7 model PC-3 model ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Blood was collected at the indicated time-points following a single dose of XL147 and plasma was prepared to determine XL147 concentrations. N = 4 mice per dose/timepoint. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 7

8 Supplemental Table S6 Efficacy of XL147 in U-87 MG, A549, A2058, and WM xenograft tumors a Xenograft Test Article Dose (mg/kg) Dosing Schedule TGI b (%) P value (vs. Veh) Regression (%) P value (vs. start) U-87 MG Vehicle - qd x XL qd x E-02 (ns) na na XL qd x E-05 na na XL Twice wkly x E-05 na na A549 Vehicle - qd x XL qd x E-03 na na XL qd x 15 > E XL Twice wkly x 5 > E (ns) A2058 Vehicle - qd x XL qd x E-07 na na WM Vehicle - qd x XL qd x E-04 na na a See Materials and Methods for details on formulation, dosing, assessment of tumor weights, and statistical analyses. See Results for details on tumor molecular alteration status. Average starting tumor size ~100 mg. b TGI, tumor growth inhibition. qd, once-daily; na, not applicable; ns, not significant. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 8

9 Supplemental Figure Legends Figure S1 XL147 inhibits PI3K pathway signaling in EGF-stimulated PC-3 cells. After incubation with XL147 at the indicated concentrations, or ZSTK474 (10 μm), PC-3 cells were stimulated with 100 ng/ml of EGF for 10 min. The cells were then lysed and effects of compound on PI3K pathway signaling assessed by western blotting. Figure S2 XL147 has no effects on nutrient-dependent mtor signaling pathway in Ramos cells. Cells were starved in serum-free media for 20 h and treated with either compound, DMSO or serum- and nutrient-free media (PBS) for 2 h. Cell lysates were prepared and analyzed by gel electrophoresis and western blotting. Cells incubated in PBS showed very low levels of phosphorylation of p70s6k, 4EBP1 or the mtor autophosphorylation site S2481. Incubation of cells in serum-free media (ie in the presence of amino acids) resulted in robust upregulation of mtor-dependent phosphorylation events. ZSTK474 and PI-103 at 10 µm inhibited these phosphorylation events, consistent with their ability to directly inhibit mtor kinase activity. Rapamycin resulted in little if any decrease in mtor autophosphorylation, but profoundly inhibited p70s6k phosphorylation consistent with selective inhibition of mtorc1. At a concentration sufficient to inhibit PI3K (100 nm) wortmannin has no effect on any of these nutrient stimulated phosphorylation events, demonstrating lack of requirement for PI3K activity. Higher concentrations caused a significant decrease at all phosphorylation sites, consistent with the ability of wortmannin to inhibit mtor at high concentrations (Peterson et al., J Biol Chem 275, , 2000). XL147 showed no effects on mtor signaling. Figure S3 XL147 inhibits endogenous PI3K pathway signaling in OVCAR-3 and U87-MG cells. After incubation for 3 hours with XL147 at the indicated concentrations, ZSTK474 (10 M), or rapamycin (0.1 M), cells were lysed and effects of compound on PI3K pathway signaling assessed by western blotting. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 9

10 Figure S4 XL147 inhibits endogenous PI3K pathway signaling in A549, MDA-MB-468, and LS174T cells. After incubation for 3 hours with XL147 at the indicated concentrations, ZSTK474 (10 M), or rapamycin (0.1 M), cells were lysed and effects of compound on PI3K pathway signaling assessed by western blotting. Figure S5 Dose-response relationships for inhibition of PI3K pathway signaling by XL147 in MCF7 and PC-3 tumors. XLfit curve fitting software (IDBS) for Excel was used to determine the doses of XL147 associated with 50% inhibition of phosphorylation at the indicated phosphoepitopes. The data used in these calculations were from the 4 h timepoints of the pharmacodynamic studies shown in Figure 4. Figure S6 Body weight plots for efficacy studies shown in Figures 5 and 6. Mice were weighed daily following initiation of administration of vehicle or the indicated agents. Values are represented as mean ± standard error. MS# MCT Pharmacodynamics and anti-tumor efficacy of XL147 (SAR245408) 10