SUPPLEMENTARY INFORMATION Materials and Methods Human cell lines and culture conditions HCC1937 is the HCC1937-pcDNA3 cell line, which was derived from a breast cancer with a mutation in exon 20 of BRCA1 (BRCA1 5382insC) and transfected with an empty pcdna3 vector [23]. HCC1937+BRCA1 is stably transfected with the BRCA1 cdna in pcdna3. EUFA423 is a BRCA2 mutant cell line derived from a Fanconi anemia (FA) brain tumor from a FANCD1 patient. This line has truncating BRCA2 mutations in exon 15 (7691 insat) of one BRCA2 allele and in exon 27 (9900 insa) of the other allele [24-28]. EUFA423+BRCA2 is stably transfected with BRCA2-HA in pcdna3. Human cell lines were maintained in 1:1 MEGM (Lonza): DMEM (Invitrogen) supplemented with 10% FBS and MEGM SingleQuots (Lonza). Cells transfected with BRCA plasmid were routinely supplemented with G418 and transferred to drug-free medium before use. Colony forming assay Cells were seeded in triplicate in 6-well plates. The following day cells were treated with drugs for 48 h (mesc) or 6 days (CHO cells). After treatment, medium was replaced with drug-free medium until colonies formed. Cells were fixed with 3:1 methanol/acetic acid, stained with 5% Giemsa stain and counted. Cell survival was calculated relative to untreated vehicle controls. Results are the mean ± standard deviation of triplicate experiments. IC 50 values were calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 5.00 for Windows (GraphPad Software). MTT cell proliferation assay 1
Cells were seeded into 96-well plates and, 24 h, later treated with indicated drug concentrations or vehicle control. At 72 h-post drug addition, a tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) was added and the resuspended formazan crystals assayed. The percentage of cell growth inhibition in triplicate experiments was expressed as [1-(A/B)]*100, where A was absorbance value for experimental wells and B for control wells. The IC 50 values were calculated as in the previous section. Cell death analysis Determination of cell death was performed with the Annexin V-FITC/Propidium Iodide (PI) staining kit (BD Pharmingen). Briefly, 72 h post-treatment, cells were collected, washed, resuspended in 1X Annexin V binding buffer and incubated (15 min, in the dark) with Annexin V-FITC and PI and 50,000 events were counted using a Cyan Flow Cytometer (Dako, USA). Image analysis was performed using Summit 4.3 software (Dako, USA). Annexin V /PI cells are viable, Annexin V+/PI are early apoptotic cells and Annexin V+/PI+ are late apoptotic and necrotic cells. The percentage of apoptotic cells was determined by adding both Annexin V+ quadrants. Western blots Cells were lysed with complete protease inhibitors cocktail (Roche Diagnostic Corporation). After centrifugation at 14000 rpm for 10 min in a microcentrifuge, supernatant was collected and protein concentrations determined using Coomassie Brilliant Blue staining (BioRad). Equal amounts of protein were separated using 4 15% Tris-HCl SDS-polyacrylamide gels, transferred onto either PVDF (BioRad) or nitrocellulose membranes (Li-Cor), and blocked using Odyssey blocking buffer (Li-Cor). Blocked membranes were incubated with the appropriate primary antibody (Anti-actin [Sigma or Cell Signaling], anti-parp [Cell Signaling], anti-caspase 3 [Cell Signaling], or anti-ercc1 [Cell Signaling]) under the recommended conditions. After washes in 1X Tris buffered saline with 0.1% Tween (TBS-T), bound primary antibodies were detected with the following secondary antibodies: AlexaFluor 680 or 800 conjugated goat anti-mouse or anti- 2
rabbit antibodies (Li-Cor Biosciences). After three washes in TBS-T, proteins were detected using an Odyssey IR Imaging System (Li-Cor Biosciences). PARP Activity Two separate experiments in duplicate or triplicate were used to determine PARP activity. Cells were grown in 10 cm cell culture dishes until they reached confluence, then washed with 0.9% NaCl, and harvested by trypsination. Cell pellets were lysed in PARP buffer. Insoluble materials were removed by centrifugation (10,000 g for 10 min at 4 o C) and proteins in the supernatant retained. Protein concentrations were determined, then proteins were flash frozen and stored at -80 o C. The assay was performed according to the recommended protocol with chemiluminescent detection performed using a Fluoroskan Ascent luminometer (Thermo Electron Corporation). A PARP linear standard curve was used to determine PARP activity for each sample. Intracellular NADPH levels 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST- 8), a water-soluble tetrazolium salt, is reduced to a yellow formazan dye by dehydrogenases, such as NADPH, in cells, allowing measurement of the amount of NADPH. Cells were plated in quintuplicate into wells of a 96- well plate and incubated 4 h at 37 o C, 5% CO 2 to allow cell attachment. Cell Counting Kit-8 solution with WST- 8 (Dojindo Molecular Technology) was added to the wells, and after incubation (4 h) absorbance (OD 450 nm) was measured. The average absorbance of the control wells was subtracted from that of the experimental wells and the relative levels of NADPH were determined. 3
Supplementary Table S1. IC 50 values for ABT-888, carboplatin, cisplatin, ABT-888/carboplatin or ABT- 888/cisplatin in different cell lines. All values are in μm. (A) Brca mesc, (B) BRCA human cancer cell lines, and (C) CHO cell lines. Note that for combination drug treatments, the IC 50 value was determined at a fixed concentration of one of the drugs, which is indicated. Also, a dash ( ) indicates that the IC 50 values were above that of the concentration range studied. A. mesc Cell Lines ABT-888 Carboplatin Cisplatin ABT-888 + 2.5 μm Carboplatin WT - 11.36 4.14-32.97 Brca1 0.87 3.40 0.34 0.15 0.28 Brca2 3.57 1.91 0.23 0.04 6.67 ABT-888 + 0.25 μm Cisplatin B. Human Cancer Cell Lines Cell Lines ABT-888 Carboplatin Cisplatin Carboplatin + 200 μm ABT-888 Cisplatin + 200 μm ABT-888 BRCA1 121.6 46.2 3.26 0.16 46.25 BRCA1 Comp. 182.1 100.1 5.91 2.17 67.92 Cell Lines ABT-888 Carboplatin Cisplatin Carboplatin + 12.5 μm ABT-888 Cisplatin + 12.5 μm ABT-888 BRCA2 13.69 12.47 1.072 3.25 2.91 BRCA2 Comp. 58.88 22.35 1.471 19.44 10.07 C. CHO Cell Lines Cell Lines Carboplatin+ ABT-888 Carboplatin ABT.0100 μm VC8 0.02 0.11.00501 VC8 + Brca2 - - - 4
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Supplementary Figure Legends SUPPLEMENTARY FIGURE S1. Clonogenic cell survival of WT and Brca mescs and inhibition of human BRCA-deficient or -complemented cell proliferation after treatment with cisplatin alone or in combination with ABT- 888. (A) Cisplatin structure. For WT (AB2.2, Brca) Brca1 (Brca1 -/- ) and Brca2 (Brca2 -/- ) mescs were treated for 48 h with the indicated drugs and concentrations and survival assessed using colony formation. (B) cisplatin and (C) ABT-888 with cisplatin. Data represent average of triplicate measurements. For (D-G), cell growth as assessed by MTT assay at 72 h for D-G post drug treatment. Percentage of growth inhibition of HCC1937 or HCC1937+BRCA1 cells after treatment with (D) cisplatin or (E) ABT-888 (200 μm)/cisplatin combination. Percentage of growth inhibition of EUFA423F and EUFA423F+BRCA2 cells after treatment with (F) cisplatin or (G) 12.5 μm ABT-888/ cisplatin combination. The percentage of cells that exhibited growth inhibition after 72 h of continuous drug treatment was compared to that of untreated cells. Data represent the average of at least triplicate samples. Error bars, mean + SEM. SUPPLEMENTARY FIGURE S2. Calculated combination index (CI) values for ABT-888/carboplatin or ABT-888/cisplatin treatments of mescs and human tumor cell lines. Data used for these plots is found in Supplementary Table 2. (a) CI values for treatment of Brca1 mescs with ABT-888 in combination with cisplatin or carboplatin. Filled circles, ABT-888/carboplatin combinations; open circles, ABT-888/cisplatin combinations. Area between the horizontal lines represents additivity, area above the upper horizontal line represents antagonism, and area below the lower horizontal line represents synergism. (b) CI values for 6
treatment of Brca2 mescs with ABT-888 in combination with either cisplatin or carboplatin. Filled circles, ABT-888/carboplatin combinations; open circles, ABT- 888/cisplatin combinations. (c) CI values for treatment of HC1937 (BRCA1- deficient) cells with ABT-888/carboplatin. (d) CI values for treatment of HC1937 (BRCA2-deficient) cells with ABT-888/carboplatin. (e) CI values for treatment of V-C8 (BRCA2-deficient) cells with ABT-888/carboplatin. SUPPLEMENTARY FIGURE S3. Clonogenic cell survival of isogenic VC8 (Brca2-deficient) and VC8+Brca2 (Brca2-complemented) CHO cells after treatment with ABT-888, carboplatin, cisplatin alone or ABT-888 combined with a platinum drug. VC8 and VC8+Brca2 cells were treated for 6 days with the indicated drugs and concentrations. (A) ABT-888, (B) carboplatin, and (C) ABT- 888/carboplatin. Data represent the average of triplicate measurements. Error bars, mean + SEM. (D) Interaction effects of ABT-888 with carboplatin treatment in VC8 CHO cell line. SUPPLEMENTARY FIGURE S4. Hematoxylin and Eosin staining (200x magnification) of tumor sections. SUPPLEMENTARY FIGURE S5. RAD51 foci formed in cells as a function of BRCA status after treatment with ABT-888/carboplatin. All RAD51 foci were analyzed by immunostaining after cells were treated with drugs for 24 h. (A) HCC1937 and HCC1937+BRCA1 treated with vehicle or 200 μm ABT-888/25 μm 7
carboplatin. (B) EUFA423 and EUFA423+BRCA2 treated with vehicle or 12.5 μm ABT-888/12.5 μm carboplatin. SUPPLEMENTARY FIGURE S6. Apoptosis in mesc after treatment with ABT-888 and carboplatin, singly or in combination. WT, Brca1, and Brca2 mescs were treated with ABT-888 (0.5 μm) and carboplatin (0.5 μm), singly or in combination. (A) Percentage of apoptotic cells after treatment with ABT- 888, carboplatin or their combination for 48 h. Apoptosis was measured by Annexin V/propidium iodide staining and analyzed by FACS. The percentage of apoptotic cells was obtained by determining the percentage of Annexin V-positive cells. (B) Western blots of Parp1 cleavage after treatment with ABT-888, carboplatin or their combination for the indicated times. The red box indicates cleaved PARP1, which is an indication of apoptosis. Actin served as a loading control. 8