Supporting Information

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 vemurafenib-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, dabrafenib, AZD6244, gefitinib, and produced in 293-GPG cells. For lentiviral infection, viruses were afatinib were purchased from Sellekchem. Erlotinib 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 kit (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”).

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