Supporting Information

Lin et al. 10.1073/pnas.1320956111 SI Methods BRAFV600E antibody was purchased from Spring Bioscience. The Cell Lines and Culture Reagents. The HCC364 cell line was kindly anti–β-actin antibody was purchased from Sigma-Aldrich. The anti- provided by David Solit (Memorial Sloan–Kettering Cancer EGFR antibody was purchased from Bethyl Laboratories. Center, New York). H1395, H1755, CAL-12T, H1666, H2405, and H2087 cell lines were purchased from American Type Cul- Plasmids, Transfection, and Viral Infection. Various pBabe–photo- ture Collection. Cells grown in RPMI 1640 supplemented with 10% activated mCherry-tagged BRAF constructs were provided by (vol/vol) FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin Xiaolin Nan (Oregon Health Sciences University, Portland, OR). were maintained at 37 °C in a humidified atmosphere at 5% CO2. The Flag-tagged or V5-tagged BRAF constructs were cloned using The HCC364 -resistant sublines VR1–VR5 were the Gateway system (Invitrogen). EGFR shRNAs were purchased maintained in the above medium and 5 μmol/L of vemurafenib. from Sigma-Aldrich. All transient transfections were conducted using Fugene 6 (Promega). For retroviral infection, viruses were Compounds. Vemurarenib, , AZD6244, , and produced in 293-GPG cells. For lentiviral infection, viruses were were purchased from Sellekchem. was pur- produced in HEK293FT cells cotransfected with packaging re- chased from LC Laboratories. agents ViraPower (Invitrogen).

Whole-Exome Sequencing. DNA from each of the indicated lines siRNA-Mediated Gene Silencing. siRNAs against EGFR and c-Jun was extracted using the QIAamp DNA mini (Qiagen). DNA were purchased from Dharmacon. BRAF siRNAs were designed (2 μg) was applied for library preparation and capture for whole- to target p61 or full-length BRAF specifically, as previously exome sequencing using Agilent SureSelect XT2 human all exon described (4). All siRNAs transfections were carried out with ’ V5 kits (Agilent) according to the manufacturer s protocols. DharmaFECT 2 Transfection Reagent at a final siRNA con- Sequencing libraries with different indices were pooled and se- centration of ∼20–50 nM, according to the manufacturer’s in- quenced in paired-end format to a length of 100 bp using the structions. About 72 h later, cells either were counted to estimate HiSeq2000 platform at the Center for Advanced Technology at cell growth or were subjected to quantitative real-time RT-PCR the University of California, San Francisco. The depth of cov- or immunoblot analysis. erage was 67–123×. Sequencing reads were aligned against Na- tional Center for Biotechnology Information build 37 (hg19) of Immunoprecipitation. The V5-tagged BRAF and FLAG-tagged – the human genome with Burrows Wheeler alignment (BWA) BRAF constructs were cotransfected into HEK293T cells. Thirty- (1). Duplicate reads were marked, local indel realignment was six hours after transfection, cells were lysed in lysis buffer (50 mM performed, and base-quality scores were recalibrated for each Tris (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA) sample with the Picard suite (http://picard.sourceforge.net/)and supplemented with protease and phosphatase inhibitor mixture the Genome Analysis Toolkit. Point mutations were identified tablets (Roche). Immunoprecipitations were performed at 4 °C for using MuTect (2); indels were identified using Pindel (3). Somatic 2 h using anti-Flag M2 magnetic beads (Sigma-Aldrich), followed mutations in the VR1– VR5 cell lines were inferred by compar- by three washes with lysis buffer. Then binding proteins were ison against the parental HCC364 cell line. eluted by FLAG-peptide and subjected to Western blot analysis. Western Blot Analysis. Cells (200,000 per well) were seeded in six- Quantitative PCR. Total RNA was collected from cultured cells well plates 24 h before drug treatment, and whole-cell lysates were using the RNeasy kit (Qiagen). cDNA was synthesized with prepared using RIPA buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl] SuperScript III reverse transcriptase using random hexamer supplemented with protease inhibitor and phosphatase inhibitor primers (Invitrogen), and RT-PCR was performed on a Quan- (Roche) and clarified by sonication and centrifugation. Equal Studio with Taqman probes (Life Technologies), using the fol- amounts of protein were separated by 4–15% SDS/PAGE and lowing program: hold at 50 °C for 2 min and polymerase acti- were transferred onto nitrocellulose membranes (Bio-Rad) for vation at 95 °C for 20 s, followed by 40 cycles of amplification GAPDH β ACTB protein blot analysis. Membranes were incubated with primary (95 °C for 1 s, 60 °C for 20 s). or -actin ( ) ex- antibody overnight and then were washed and incubated with pression was used as an internal reference to normalize input secondary antibody. Membranes were exposed using either a cDNA. Then ratios of the expression level of each gene to that of fluorescence system (LI-COR) or a chemiluminescent reagent; the reference gene were calculated. images were captured, and bands were quantified using an Im- ageQuant LAS 4000 instrument (GE Healthcare). Transformation Assay. Soft agar assays were performed according to published methods (5). Stable clones of NIH 3T3 cells infected Antibodies. Anti–phospho-MEK, anti–phospho-ERK, anti–phospho- by different BRAF constructs were plated onto DMEM soft agar EGFR, anti–phospho-AKT, anti–phospho-JNK, anti–phospho-Jun, plates at a density of 10,000 cells per well in six-well plates and anti-AKT, anti-JNK, anti-Jun, and anti-Bim antibodies were were treated with different concentrations of vemurafenib (me- purchased from Cell Signaling Technology. The anti-BRAF kinase dium was changed every 3 d). Plates were incubated for 3 wk and antibody was purchased from Santa Cruz Biotechnology. The anti- examined by microscopy to quantify colonies.

1. Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler 4. Poulikakos PI, et al. (2011) RAF inhibitor resistance is mediated by dimerization of transform. Bioinformatics 26(5):589–595. aberrantly spliced BRAF(V600E). Nature 480(7377):387–390. 2. Cibulskis K, et al. (2013) Sensitive detection of somatic point mutations in impure and 5. Clark GJ, Cox AD, Graham SM, Der CJ (1995) Biological assays for Ras transformation. heterogeneous cancer samples. Nat Biotechnol 31(3):213–219. Methods Enzymol 255:395–412. 3. Ye K, Schulz MH, Long Q, Apweiler R, Ning Z (2009) Pindel: A pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25(21):2865–2871.

Lin et al. www.pnas.org/cgi/content/short/1320956111 1of10 A

Vemurafenib Dabrafenib

1.5 1.5

1.0 1.0

0.5 0.5 Relative cell viability Relative cell viability 0.0 0.0 0.001 0.01 0.1 1 10 00.010.11 10 Conc (μM) Conc (μM)

AZD6244 V600E HCC364 BRAF 1.5 H1755 BRAF G469A H1395 BRAF G469A L485_P490Y 1.0 H2405 BRAF G466V CAL-12T BRAF G466V 0.5 H1666 BRAF H2087 BRAF L597V Relative cell viability 0.0 0.001 0.01 0.1 1 10 Conc (μM)

B

Vemurafenib 2 μM -+-+-+ -+ -+ -+-+ pMEK

pERK

Actin

HCC364 CAL12T H1666 H1395 H1755 H2405 H2087 BRAF V600E BRAFG466V BRAF G466V BRAFG469ABRAF G469A BRAF L485_P490Y BRAF L597V

C HCC364 BRAFV600E 1.4

1.2

1.0 Parental VR1 0.8 VR2 0.6 VR3 VR4 0.4 VR5

Relative cell viability 0.2

0.0 0 0.01 0.1 1 10 Dabrafenib (μM)

Fig. S1. Response of NSCLC cell lines harboring various BRAF mutations to the BRAF inhibitors vemurafenib and dabrafenib and the MEK inhibitor AZD6244 (see Fig. 1). (A) Dose–response curve showing the effect of vemurafenib, dabrafenib, and AZD6244 on the viability of NSCLC cell lines harboring different BRAF mutations. H2087 cells also harbor an NRAS Q61K-activating mutation. (B) Western blot analysis of each indicated protein in lysates from HCC364, CAL12T, H1666, H1395, H1755, H2405, and H2087 cells. (C) Dose–response curve showing the effect of dabrafenib on the viability of the HCC364 parental cell line and the five derived vemurafenib-resistant lines. Data shown (±SEM) are normalized to DMSO treatment (n = 3).

Lin et al. www.pnas.org/cgi/content/short/1320956111 2of10 A B

V600E VR1 BRAFVE 1 2 3 4 5 6 7 8 9101112131415161718 VR2 V600E VR3 p61VE VR4 VR5

C D p61 cDNA cloned from VR1 HCC364 V600E exon 3 exon 9

Parental VR1 VR2 VR3

2.3Kb FL-BRAF

1.7Kb p61

E F AR 1-5 Parental VR1 VR1 off-drug (2 months) Vemurafenib Parental -+-+ -+ 2 μM tBraf BRAF Pan- p61 BRAF BRAFVE VE- BRAF p61VE Actin pMEK HCC364 Actin G (A+V)R 1-5 Parental tBraf

Actin HCC364

Fig. S2. Detection of p61VE in VR1 cells (see Fig. 2). (A) Dendrogram showing unsupervised clustering of VR1– VR 5 sublines based on transcriptome data. (B) Schematic exonic structure of the BRAFV600E full-length gene and the BRAFp61VE splicing variant. (C) PCR amplification of full-length BRAF and the BRAF p61 variant from cDNA of parental HCC364, VR1, VR2, and VR3 cells. (D) Snapshot of Sanger sequencing results of a p61VE variant from cDNA of VR1 cells showing a V600E mutation and a junction between exon 3 and exon 9. (E) Western blotting to detect full-length BRAF, p61VE, and pMEK in cell lysates from HCC364 parental cells, VR1 cells, and VR1 cells grown for 2 mo without vemurafenib in the culture medium. (F) Western blotting to detect BRAF expression in cell lysates from five distinct HCC364 subclones with acquired resistance to the MEK inhibitor AZD6244 (denoted “A”). (G) Western blotting to detect BRAF in cell lysates from five distinct HCC364 subclones with acquired resistance to the combination of the MEK inhibitor AZD6244 (denoted “A”) and vemurafenib (denoted “V”, hence “A+V”).

Lin et al. www.pnas.org/cgi/content/short/1320956111 3of10 NIH3T3 A B EV WT VE VEHp61VEH p61VE C WT VE VEH p61VE p61VEH Vemurafenib -+-+-+ -+- + -+ IP: FLAG 1μM IB: V5 mCherry

tBRAF IP: FLAG IB: FLAG pMEK

pERK Input IB: V5 Actin

Input IB: FLAG

C NIH3T3 EV 40 BRAF WT BRAF VE 30 * * BRAFp61VE * * BRAFp61VEH 20

10 Number of Colonies

0

5μM 1μM 2μM DMSO Vemurafenib

DEHCC364 HCC364 4 p<0.01 p<0.01 EV BRAFVE BRAFVEH BRAFp61VE BRAFp61VEH Vemurafenib 0.5125 0.5125 0.51250.5125 0.5125 3 μM mCherry 2 tBRAF 1 pMEK

Relative cell number cell Relative 0 pERK

pBabe p61 VE Actin BRAF VE p61 VEH BRAF WT BRAF VEH pMEK-Red Vemurafenib (5 μM) pERK-Green

Fig. S3. A dimerization-impaired mutant of p61VE exhibits enhanced sensitivity to vemurafenib (see Fig. 2). (A) Immunoprecipitation (IP) analysis to compare the levels of dimerization among different BRAF proteins. HEK293T cells cotransfected with Flag-tagged/V5-tagged BRAF-WT, BRAF-V600E (BRAF-VE), BRAF- V600E-R509H (BRAF-VEH), p61VE, and p61BRAF-V600E-R509H (p61VEH) constructs were lysed followed by immunoprecipitation with FLAG antibody. Then Western blotting using anti-FLAG or anti-V5 antibodies was performed as indicated. (B) Western blot analysis to compare MEK/ERK activation and sensitivity to vemurafenib in NIH 3T3 cells stably expressing mCherry-tagged BRAF-WT, BRAF-VE, BRAF-VEH, p61VE, and p61VEH proteins. (C) NIH 3T3 transformation assay to compare transformation capacities and sensitivities to vemurafenib in NIH 3T3 cells stably expressing mCherry-tagged BRAF-WT, BRAF-VE, p61VE, and p61VEH proteins. (D) Long-term growth assay (10 d) to compare sensitivity to vemurafenib in HCC364 parental cell lines stably expressing mCherry-tagged BRAF-WT, BRAF-VE, BRAF-VEH, p61VE, and p61VEH proteins. (E) Western blot analysis to compare MEK/ERK activation and sensitivity to vemurafenib in HCC364 parental cells stably expressing mCherry-tagged BRAF-VE, BRAF-VEH, p61VE, and p61VEH proteins.

Lin et al. www.pnas.org/cgi/content/short/1320956111 4of10 EGFR Parental

R3

R4

R5

Fig. S4. Phospho-receptor (RTK) array showing RTK activity in the parental HCC364 cell line and in the VR3, VR4, and VR5 sublines. The data reveal no significant up-regulation of pEGFR (or other RTKs) in the VR3, VR4, or VR5 vemurafenib-resistant subclones.

Lin et al. www.pnas.org/cgi/content/short/1320956111 5of10 A BC HCC364 VR3 HCC364 VR3 HCC364 VR3 1.2 1.4 1.2

1.2 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 P-Vemurafenib 0.4 0.4 P-Vemurafenib P-Vemurafenib VR3-Vemurafenib 0.4 VR3-Vemurafenib VR3-Vemurafenib Relative Cell Vilability 0.2 VR3-Vemurafenib Relative Cell Vilability 0.2

Relative Cell Vilability VR3-Vemurafenib +5 μM Gefitinib VR3-Vemurafenib 0.2 0.0 +1 μM Afatinib +100 nM Afatinib 0.0 00.11 10 0.0 0 0.1 1 10 0 0.1 1 10 Vemurafenib (μM) Vemurafenib (μM) Vemurafenib (μM)

D E HCC364 VR4 1.4 HCC364 VR4 Erlotinib 5 μM - + - + 1.2 Vemurafenib 2 μM --++ pEGFRY1068 1.0

0.8 P-Vemurafenib tEGFR pMEK 0.6 VR4-Vemurafenib VR4-Vemurafenib pERK 0.4 +5 μM Erlotinib BIM 0.2 Relative viability to control to viability Relative

0.0 pAKT 00.1110 Actin Vemurafenib (μM)

F G HCC364 VR4 HCC364 VR4 1.2 1.2

1.0 1.0 P-Vemurafenib P-Vemurafenib 0.8 0.8 VR4-Vemurafenib VR4-Vemurafenib 0.6 0.6 VR4-Vemurafenib 0.4 VR4-Vemurafenib 0.4 +5 μM Gefitinib +1 μM Afatinib 0.2 0.2 Relative Cell Vilability Relative Cell Vilability

0.0 0.0 00.1110 00.11 10 Vemurafenib (μM) Vemurafenib (μM)

Fig. S5. The EGFR inhibitors erlotinib, gefitinib, and afatinib overcome resistance to vemurafenib in HCC364 VR3 and VR4 cells (see Fig. 4). (A) Dose–response curves showing the effect of each indicated inhibitor (vemurafenib, gefitinib, or combined) on the viability of the indicated signaling components in HCC364 parental line and VR3 cells. (B) Dose–response curve showing the effect of each indicated inhibitor (vemurafenib, afatinib, or combined) on the viability of the indicated signaling components in the HCC364 parental cell line and VR3 cells. (C) Dose–response curve showing the effect of each indicated inhibitor (vemurafenib, afatinib, or combined) on the viability of the indicated signaling components in the HCC364 parental line and VR3 cells. (D) Dose–response curve showing the effect of each indicated inhibitor (vemurafenib, erlotinib, or combined) on the viability of the indicated signaling components in the HCC364 parental cell line and VR4 cells. (E) Western blot analysis of each indicated protein in lysates from the VR4 line treated with vemurafenib, erlotinib, or the combination as indicated. (F) Dose–response curve showing the effect of each indicated inhibitor (vemurafenib, gefitinib, or combined) on the viability of the indicated signaling components in the HCC364 parental cell line and VR4 cells. (G) Dose–response curve showing the effect of each indicated inhibitor (vemurafenib, afatinib, or combined) on the viability of the indicated signaling components in the HCC364 parental cell line and VR4 cells. Data shown(±SEM) are normalized to DMSO treatment (n = 3).

Lin et al. www.pnas.org/cgi/content/short/1320956111 6of10 A B HCC364 HCC364 VR3 Vemurafenib 2μM - ++- - ++- - ++- ** ** (6 hours) 1.5 EGFR siRNA - - + + - - + + - - + + pEGFR

1.0 tEGFR pMEK 0.5 pERK Relative cell viability 0.0 pAKT DMSO 1 μM 2 μM Vemurafenib Actin Parental VR3 VR4 siRNA control HCC364 VR4 EGFR siRNA 1.5 ** ** C

1.0 HCC364 VR4 1.2

0.5 1.0 Relative cell viability 0.8 0.0 DMSO 1 μM 2 μM Vemurafenib 0.6

0.4 Relative cell viability 0.2 shRNA control EGFR shRNA 0.0 0 1 10 20 Vemurafenib (μM)

Fig. S6. Genetic abrogation of EGFR overcomes resistance to vemurafenib in HCC364 VR3 and VR4 cells (see Fig. 4). (A) Effects of siRNAs against EGFR in VR3 (Upper) and VR4 (Lower) cells with and without vemurafenib treatment. (B) Western blot analysis of each indicated protein in lysates from the HCC364 pa- rental cell line and from VR3 and VR4 cells transfected with siRNA (control) or EGFR siRNAs treated with and without vemurafenib (6 h) as indicated. (C) Dose– response curve showing the effect of vemurafenib on the viability of VR4 cells infected with a control or EGFR-directed shRNA. Data shown (±SEM) are normalized to DMSO treatment (n = 3).

Lin et al. www.pnas.org/cgi/content/short/1320956111 7of10 A B HCC364 HCC364 0.20 BRAF BRAF Control siRNA-1 siRNA-2 siRNA Vemurafenib 0.15 2 μM -+-- + + BRAF 0.10

pEGFR 0.05

tEGFR 0.00 Normaized mRNA expression Actin BTC EGF TGF-α AREG EREG HB-EGF

C

1.2 TGFA 1.2 AREG 1.2 EREG 1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4 TGFA/GAPDH EREG/GAPDH AREG/GAPDH 0.2 0.2 0.2

0.0 0.0 0.0 2 l

siRNA-

siRNA control BRAF siRNA-1 BRAF siRNA contro BRAF siRNA-1 BRAF siRNA-2 siRNA control BRAF siRNA-1 BRAF siRNA-2

Fig. S7. EGFR ligand expression and EGFR activation are positively regulated by BRAF signaling (see Fig. 5). (A) Western blot analysis shows that genetic abrogation of BRAF in the HCC364 parental cell line by siRNA significantly decreases phosphorylation of EGFR. (B) The EGFR ligands TGF-α, AREG, EREG, and HB-EGF are highly expressed in HCC364 parental cells. BTC, . (C) Quantitative RT-PCR results showing that genetic abrogation of BRAF by siRNA in HCC364 parental cells significantly decreases transcription of the EGFR ligands TGF-α, AREG, and EREG.

Patient #1 Patient #2

Pre-treatment

Acquired dabrafenib resistance

pS6 S235/236

Fig. S8. Immunohistochemistry staining of pS6 S235/236 in pretreatment and acquired-resistance biopsies from two patients with BRAFV600E NSCLC who were treated with the BRAF inhibitor dabrafenib. The data reveal increased levels of pS6 in the tumor cells in the specimen from patient #2 with acquired dabrafenib resistance (Lower Right) in which increased pAKT levels also were observed (Fig. 6B), compared with the pretreatment matched tumor specimen from this patient. Arrows indicate pS6+ tumor cells. (Scale bars, 50 μM.)

Lin et al. www.pnas.org/cgi/content/short/1320956111 8of10 Table S1. Effects of each BRAF inhibitor on the human NSCLC cell lines (also see Fig. 1A)

Cell line Vemurafenib IC50, μM Dabrafenib IC50, μM

HCC364 Parental 0.75 ± 0.05 0.1 ± 0.02 VR1 >10 >10 VR2 >10 >10 VR3 >10 >10 VR4 >10 >10 VR5 >10 >10

Listed are IC50 values of parental HCC364 and VR1–VR5 cell lines treated with the BRAF inhibitors vemurafenib or dabrafenib. IC50 values (±SEM) were derived from cell-viability assays after 72 h of drug treatment (n = 3).

Table S2. Synergy score for each targeted agent in combination with vemurafenib in the HCC364 VR3 cells (also see Fig. 4B) Drug Synergy score Standard deviation

Gefitinib(Iressa)_EGFR_RTK 3.5611 0.9396 AZD1152(Barasertib)_AuroraKinase_DNArepair 3.0876 1.274 ErlotinibHydrochloride_EGFR_RTK 3.0307 1.0273 17-AAG(Geldanamycin)_HSP_OTHER 2.0743 1.9689 AZD7762_CHK_DNArepair 1.7432 0.1723 CX-4945_PKC_KINASE 1.2467 0.0446 A66_PI3K_PI3K/AKT 1.2263 0.8377 AZD2281(Olaparib)_PARP_DNArepair 1.1984 0.039 CI-1033(Canertinib)_EGFR,HER2_RTK 1.1851 0.4658 XL880(GSK1363089)_c-Met,VEGFR_RTK 1.1328 0.2529 BEZ235(NVP-BEZ235)_mTOR,PI3K_TORC 1.1308 1.2038 SB431542_ALK_RTK 1.0798 0.3787 CP-724714_HER_RTK 1.0356 0.6302 PLX-4720_B-Raf_KINASE 0.8268 1.2385 Tipifarnib_farnesyltransferase(Ftase)_OTHER 0.8256 0.4501 OSI-930_c-Kit,VEGFR_RTK 0.7331 0.3871 MP-470_c-Met,c-Kit,PDGFR,Flt_RTK 0.7084 0.1077 Vandetanib_VEGFR,EGFR,RET_RTK 0.6757 1.3579 BI2536_PLK_KINASE 0.6506 0.0383 SunitinibMalate_VEGFR,PDGFR,c-Kit,Flt_RTK 0.5964 0.0475 S31-201_STAT_KINASE 0.5902 0.4314 Abiraterone(CB-7598)_P450_OTHER 0.5891 0.4695 BI6727_PLK_KINASE 0.4534 0.2867 BIIB021_HSP_OTHER 0.4282 0.2612 YM155_Survivin_Apoptosis 0.4199 0.1896 LapatinibDitosylate_EGFR,HER2_RTK 0.3906 0.641 ABT-888_PARP_DNArepair 0.3835 0.7474 Adriamycin_Topoisomerase_Chemo 0.3488 0.51 LDE225_Smo_HEDGEHOG 0.2703 0.2709 Cyclopamine_Hedgehog_HEDGEHOG 0.2324 0.4807 Etoposide_Topoisomerase_Chemo 0.2261 0.1281 GDC-0941_PI3K_PI3K/AKT 0.2068 0.457 PD0325901_MEK_MEK 0.2056 0.4072 SB743921_Ksp_KINASE 0.2013 0.5176 PIK-90_PI3K_PI3K/AKT 0.1686 1.157 AZD6244()_MEK_MEK 0.1365 2.3828 PIK-75Hydrochloride_PI3K_PI3K/AKT 0.0992 0.0655 GemcitabineHydrochloride_Antimetabolites_Chemo 0.097 1.4482 OSU-03012_PDK_KINASE 0.0854 0.1176 LY2603618_CHK_DNArepair 0.0773 0.4604 AC-220_FLT_RTK 0.0653 0.3434 (STI571)__RTK 0.0638 1.5662 Perifosine_Akt,PI3K_PI3K/AKT 0.0454 0.7201 LY2157299_TGF-beta_OTHER 0.0409 2.1696 NVP-TAE684_ALK_RTK 0.0409 0.4043 PI-103_DNA-PK,PI3K,mTOR_KINASE 0.0212 0.0825

Lin et al. www.pnas.org/cgi/content/short/1320956111 9of10 Table S2. Cont. Drug Synergy score Standard deviation

Cytarabine__Chemo −0.0077 0.2565 Tandutinib(MLN518)_Flt_RTK −0.02 0.3165 Cisplatin__Chemo −0.0255 0.1345 AZD0530(Saracatinib)_SRC,Bcr-Abl_RTK −0.0439 2.7123 VX-680_AuroraKinase_KINASE’ −0.1554 0.2025 SNS-032(BMS-387032)_CDK_KINASE −0.172 0.0176 Nutlin-3_MDM2_OTHER −0.1921 0.4391 XAV-939_Wnt_OTHER −0.2137 0.1183 SGI-1776_Pim_KINASE −0.2914 0.4848 LY294002_PI3K_PI3K/AKT −0.347 0.3585 Vorinostat_HDAC_HDAC −0.3607 0.5144 PP242_mTOR_TORC −0.3654 0.3703 JNJ26854165_p53_OTHER −0.3823 0.8108 MK-1775_Wee1_KINASE −0.405 0.5486 XL184_c-Met/FLT-3_RTK −0.4616 1.4823 SU11274(PKI-SU11274)_c-Met_RTK −0.4921 0.7028 GDC-0449(Vismodegib)_Hedgehog,P-gp,ABC_HEDGEHOG −0.5097 0.3626 RO4929097_Y-Secretase_OTHER −0.5257 0.058 LDN193189_ALK_RTK −0.5449 0.8686 CHIR-99021_GSK-3_KINASE −0.5639 0.2423 BIBW2992(Afatinib)_EGFR,HER2_RTK −0.6006 0.6504 Flavopiridol_CDK_KINASE −0.6109 0.4862 SB216763_GSK-3_KINASE −0.7402 0.2502 PLX-4032_B-Raf_KINASE −0.7553 0.8234 CI-1040(PD184352)_MEK_MEK −0.7885 2.3663 TGX-221_PI3K_PI3K/AKT −0.8406 0.0712 Everolimus(RAD001)_mTOR_TORC −0.8799 1.079 Paclitaxel(Taxol)_MicrotubuleFormation_Chemo −0.9029 1.0944 Lenalidomide_TNF-alpha_Apoptosis −0.9549 0.7783 GSK429286A_ROCK_KINASE −1.1369 0.1698 R935788(Fostamatinibdisodium)_Syk_RTK −1.1894 0.5213 KU-55933_ATM_DNArepair −1.3788 0.331 TrichostatinA_HDAC_HDAC −1.4128 0.0175 JNJ-38877605_c-Met_RTK −1.5071 0.8048 FasudilHCl_ROCK_KINASE −1.5177 0.2093 ABT-263(Navitoclax)_Bcl-2_Apoptosis −1.5227 1.3961 CCT128930_Akt_PI3K/AKT −1.5921 1.8973 MK-2206_Akt_PI3K/AKT −1.7044 1.7253 Neratinib_HER2_RTK −1.7711 0.879 Dasatinib_SRC,Bcr-Abl,c-Kit_RTK −2.1102 0.8958 PF-2341066_c-Met,ALK_RTK −2.2182 1.2345 MG132_Proteasome_OTHER −2.4651 0.8822 GSK690693_Akt,AGCKinase_PI3K/AK −2.5314 1.2224 ’Bortezomib_Proteasome_OTHER −3.6572 2.857

Listed are pharmacological compounds used in the chemical screen ranked by synergy score along with their primary targets. The synergy scores shown are the average synergy scores calculated over a dose–response curve for each drug in the library (doses of 5 μg/mL, 500 ng/mL, and 50 ng/ mL).

Lin et al. www.pnas.org/cgi/content/short/1320956111 10 of 10