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1 Supporting Information Johnson et al /pnas SI Materials and Methods Cell Lines and Reagents. Cell lines were obtained from the American Type Culture Collection. Puromycin, blasticidin, DMSO, MG132, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17- DMAG) were purchased from Sigma-Aldrich, bortezomib from Selleck, and cisplatin from Roche Pharmaceuticals. Clovis provided rucaparib (PF ), and AstraZeneca provided olaparib (AZD2281). Derivation of Resistant Clones. MDA-MB-436 cells were cultured in the presence of 30 nm rucaparib in six-well plates until resistant clones emerged ( 2 mo). Rucaparib-resistant (RR) clones RR-1 to RR-6 were subjected to gradual increases in rucaparib concentrations until cells could grow in the presence of 1 μm ( 4 6 mo from initial exposure). Cells were cultured in the absence of rucaparib a minimum of 1 mo before they were used for experiments. Colony Formation Assays. MDA-MB-436 parental and resistant cells were seeded in six-well plates at 1,000 cells per well in the presence of increasing concentrations of rucaparib or olaparib. Resistant clones were also cultured in the absence of rucaparib for 6 mo and assessed by colony formation in the presence of rucaparib. For cisplatin treatments, exponentially growing cells were cultured in 24-well plates, treated with increasing concentrations of cisplatin for 24 h, and replated at 1,000 cells per well in six-well plates for colony formation. Colony formation was assessed 2 wk after plating with crystal violet staining. For shrna or cdna add-back colony formation experiments, cells were treated as described earlier, but with the addition of puromycin or blasticidin in the media, respectively. For sirna treatments, exponentially growing cells were reverse-transfected in 24-well plates, and 2 d posttransfection cells were treated with rucaparib for 72 h and then replated in six-well plates for colony formation. For 17-DMAG colony assays, cells were treated with 17-DMAG (50 nm) and rucaparib (100 nm) for 72 h and then replated into six-well plates for colony formation. Colony formation was assessed 2 wk after plating with crystal violet staining. Mean colony formation from three experiments was expressed as percentage of colonies ± SE relative to vehicletreated cells. Immunoprecipitation, Western Blotting, Immunofluorescence, and Focal Microscopy. BRCA1 (Calbiochem) and HSP90 (Enzo Life Sciences) antibodies were used for immunoprecipitation of BRCA1 or HSP90 complexes from 2 mg of nuclear extract by using a Pierce Classic IP Kit (Thermo Scientific) according to the manufacturer s instructions. Nuclear extracts were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer s instructions. Separation of soluble and chromatin extracts was performed as previously described (1). Western blotting was carried out as previously described, and proteins were detected by using the following antibodies: poly(adp-ribose) (PAR) (GeneTex); poly(adp-ribose) polymerase (PARP)-1 (Cell Signaling); histone H3 (Cell Signaling); tubulin (Cell Signaling); 53BP1 (Cell Signaling); RAD51 (Santa Cruz); N-terminal BRCA1 (Calbiochem); C-terminal BRCA1 (Santa Cruz); ORC2 (Santa Cruz); MCL-1 (Cell Signaling), HSP90 (Enzo Life Sciences); HSP70 (Cell Signaling); CtIP (Bethyl Labs); PARP-2 (Millipore); BARD1 (Santa Cruz); PALB2 (Bethyl Labs); BRCA2 (Calbiochem); and RAP80 (Bethyl Labs). Densitometric analyses were carried out by using ImageJ 1.45 software (National Institutes of Health). For immunofluorescence, cells were routinely analyzed 6 h after 10 Gy γ-irradiation treatment. Preparation of cells and methods have been described previously (2). For metaphase spreads, we treated cells for 24 h in the presence of 1 μm rucaparib and then harvested, and cells were counted as described previously (2). RNAi and cdna Add-Back Treatments. We purchased Hs_BRCA1_ FlexiTube sirna constructs 1 to 3 (construct 2, SI ; construct 9, SI ; construct 13, SI ), and AllStars Negative Control sirna (scrambled control) from Qiagen. CtIP sirna (Set of 4: ON-TARGETplus RBBP8), 53BP1 sirna (SMARTpool: ON-TARGETplus TP53BP1), as well as PARP-1 and PARP-2 sirnas, were purchased from Dharmacon. Bioinformatic design of sirna specific to the exon 20 deletion variant resulted in the sequence 5 - AAATGCTGAATGAGATCTT-3, and the insertion intron 20 variant resulted in the sequences (i) 5 -TGCAAAGGGGAGTGGAATA-3, (ii) 5 - GGGAGGGAG- CTTTACCTTT-3, and(iii) 5 - GGGGAGTGGAATACAGAGT- 3. Transfections were carried out according to standard protocols. 53BP1 TP53BP1 cdna pdonr223 Gateway Entry vector was purchased from Open Biosystems and shuttled into plx304 Gateway Destination vector (Addgene). GFP cdna-plx304 was a gift from David Root (Broad Institute, Cambridge, MA). Lentiviral generation and infections were carried out according to standard protocols. Protein knockdown or reexpression was routinely assessed 72 h after transfection or 96 h after infection. Cells infected with shrna or cdnas were maintained in 3 μg/ml puromycin or blasticidin, respectively, to generate stable cell lines. Gene Sequencing, SNP Array Analyses, and Quantitative RT-PCR. Genomic DNA was isolated from cells by using the DNeasy tissue kit (Qiagen). BRCA1 gene sequencing was carried out as previously described (3). Initial DNA sequencing of TP53BP1 was performed by using targeted capture and massively parallel genomic sequencing (4). Validation of the TP53BP1 indel was performed by Sanger sequencing using PCR primers to amplify exon 18. PCR amplicons were sequenced bidirectionally using Applied Biosystems Big Dye Terminator v3.1 Cycle Sequencing Kit and an ABI 3130xl genetic analyzer. Trace sequences were analyzed using Sequencher v4.9 software (Gene Codes) and ABI Sequence Scanner v1.0 software. SNP chip array was carried out using the human SNP 6.0 array according to the manufacturer s instructions (Affymetrix). Comparison of gene copy number differences between sensitive and resistant cells was performed using the dchip software according to previously established methods (5). Total RNA was isolated from cell lines using TRIzol (Invitrogen) and purified using RNeasy cleanup kit (Qiagen). BRCA1 mrna levels were measured as previously described (6). Assessment of BRCA1 transcript variants was carried out by using primers specifictoexon15and exon 21 as previously described (7). Comparison of DNA sequences was carried out by using the Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology Information). Histologic and Immunohistochemical Staining. Formalin-fixed, paraffin-embedded sections were obtained from primary and recurrent ovarian carcinomas from women with BRCA1 germ-line mutations after informed consent on one of two institutional review board (IRB)-approved protocols (University of Washington 1of8

2 IRB 27077, and Fred Hutchinson Cancer Research Center IRB 7016). Immunohistochemistry was performed as previously described (3) by using primary 53BP1 antibody diluted 1:20 (gift from Thanos Halazonetis, University of Geneva, Geneva, Switzerland) and BRCA1 antibody diluted 1:200 (Calbiochem). Staining was assessed by using standard quantitative scoring methods, i.e., 1+, 2+, 3+, 4+. Increase or decrease in staining indicatedintables1wasdetermined according to the change in quantitative scoring. Statistical Analyses. Mean and SE values were compared by using unpaired t tests or, for multiple comparisons, one-way ANOVA (GraphPad Software). P < 0.05 was considered statistically significant. 1. Johnson N, et al. (2009) Cdk1 participates in BRCA1-dependent S phase checkpoint control in response to DNA damage. Mol Cell 35(3): Johnson N, et al. (2011) Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition. Nat Med 17(7): Swisher EM, et al. (2008) Secondary BRCA1 mutations in BRCA1-mutated ovarian carcinomas with platinum resistance. Cancer Res 68(8): Walsh T, et al. (2011) Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci USA108(44): Zhao X, et al. (2005) Homozygous deletions and chromosome amplifications in human lung carcinomas revealed by single nucleotide polymorphism array analysis. Cancer Res 65(13): Moskwa P, et al. (2011) mir-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Mol Cell 41(2): Bonnet C, et al. (2008) Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet 45(7): Fig. S1. (A) To measure PARP activity, PAR levels were measured in cell extracts by Western blot by using PAR-specific antibodies in the absence of activated DNA. MDA-MB-436 parental cells and resistant clones were treated with rucaparib for 24 h, and PAR, PARP-1, and tubulin levels were measured by Western blot. All clones treated with 1 μm rucaparib had reduced PAR levels with the exception of RR-1 cells, in which the degree of reduction was substantially less. RR-5 and RR-6 had lower basal PAR levels. (B) We investigated the possibility that the primary reason for drug resistance in RR-1 cells was the inability of rucaparib to inhibit PARP and reduce PAR levels. Because rucaparib is an inhibitor of PARP-1 and PARP-2, we transfected MDA-MB-436 parental, RR-1, and RR-5 cells with 20 nm scrambled (Sc), PARP-1 (P1), PARP-2 (P2), or PARP-1 and PARP-2 (1+2) sirna, and PAR, PARP-1, PARP-2, and tubulin levels were measured by Western blot. In MDA-MB-436 parental cells, PARP-1 sirna reduced PAR levels; however, PARP-2 sirna had no effect on PAR levels. Interestingly, PARP-1 and PARP-2 sirna both reduced PAR levels in RR-1 cells. RR-5 cells had low basal PAR levels. (C) Cells were transfected as in B, and 72 h posttransfection cells were reseeded and colony formation assessed 2 wk later. PARP-1 sirna reduced colony formation in MDA-MB-436 parental cells. Despite PARP-1 and PARP-2 sirna reducing PAR levels in RR-1 cells, colony formation was only slightly impacted. RR-5 cells have lower overall PAR levels, and PARP-1 or PARP-2 sirna did not affect colony formation. 2of8

3 Fig. S2. (A) Genomic DNA was extracted from MCF7 cells, MDA-MB-436 parental cells and resistant clones and BRCA1 introns and exons were subjected to Sanger sequencing; representative electropherograms surrounding the G>A mutation are depicted. (B) Analyses ofbrca1 mrna by RT-PCR. DNA gel demonstrating RT-PCR products obtained from MCF7, MDA-MB-436 parental cells and resistant clones using primers spanning exons 15 to 21 (1). MCF7 cells are WT for the BRCA1 gene, and one transcript is detectable. In contrast, MDA-MB-436 parental and resistant clones contain G>A mutation in the splice donor site of exon 20. Consequently, two transcripts are produced. The first contains an in-frame deletion of exon 20, resulting in a band lower than the MCF7 WT BRCA1 band; the other transcript retains intron 20, resulting in a band higher than the WT BRCA1 band (2). There is no change in the size of the alternatively spliced transcripts from MDA-MB- 436 parental and resistant clones. (C) Bands detected by agarose gel electrophoresis in B were gel-extracted, purified, and sequenced. Images show DNA sequences Legend continued on following page 3of8

4 from a BLAST query of parental cells vs. clone RR-1. The BRCA1 sequences from parental cells and the resistant clone were identical for the insertion intron 20 variant (Left) and the deletion exon 20 variant(right). (D) The BRCA G>A mutation present in MDA-MB-436 parental cells and RR clones produces two transcript variants. MDA-MB-436+WT cells and resistant clone RR-6 were treated with sirna specifically targeting insertion intron 20 or deletion exon 20 BRCA1 variants. Bioinformatic sirna design provided three sirnas targeting the insertion of intron 20 variant and one sirna targeting the deletion of exon 20 variant. (Top Left) Depiction of the WT BRCA1 gene, mrna, and protein. Exons 16 to 22 are highlighted. (Top Right) BRCA1 WT mrna sequence from exon 21 (blue) to 20 (green). (Middle Left) BRCA G>A variant 1 mrna and protein are depicted. Insertion of intron 20 results in a stop codon and truncated protein. Bioinformatic sirna design provided three sirnas that specifically targeted BRCA1 mrna from the insertion of intron 20 variant over WT or variant 2. (Middle Right) mrna sequences of variant 1 are shown from exon 21 (blue) to exon 20 (green) and the insertion of intron 20 (yellow). sirnas and corresponding targeted mrna regions are demonstrated. (Bottom Left) BRCA G>A variant 2 mrna and protein are depicted. An in-frame deletion of exon 20 results in a truncated protein lacking amino acids coded by exon 20. (Bottom Right) Bioinformatic sirna design provided one sirna that specifically targeted the mrna from the deletion of exon 20 variant over WT or variant 1. Exon 19 (pink) and exon 21 (blue) mrna sequences are shown with sirna corresponding to the mrna region targeted. (E) MDA-MB-436+WT cells and resistant clone RR-6 were treated with sirna specifically targeting insertion intron 20 or deletion exon 20 BRCA1 variants. Western blots demonstrate that BRCA1 is reduced in resistant clone RR- 6 using deletion exon 20 sirna but WT BRCA1 levels are not reduced. These data suggest that the exon 20 deletion variant accounts for the predominant variant overexpressed in resistant clones. 1. Bonnet C, et al. (2008) Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet 45(7): Elstrodt F, et al. (2006) BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. Cancer Res 66(1): Fig. S3. MDA-MB-436 parental cells and resistant clones RR-1, RR-5, and RR-6 were treated as in Fig. 2B and assessed for soluble (marked as s ) and chromatin-bound (marked as p ) BRCA1, RAD51, ORC2 and tubulin protein levels by Western blot. Fig. S4. (A) MDA-MB-436 parental cells (436) and resistant clones RR-1, RR-5, and RR-6 were assessed for gene copy number by SNP chip array analyses. Copy number for SNPs at chromosome 17 are displayed. Intensity of red indicates increase or decrease in copy number (Left); blue bar passing the red line indicates increased copy number (Right). (B) BRCA1 mrna levels normalized to GAPDH from MDA-MB-436 parental and resistant clones were measured by quantitative RT-PCR. For each experiment, the normalized BRCA1 mrna values from each resistant cell line were divided by the value obtained for MDA-MB-436 parental cells to derive a fold change value, whereby, in each experiment, the value in MDA-MB-436 cells is equal to 1 (n = 3, mean ± SD; *P < 0.05, one-way ANOVA). (C) MDA-MB-436 parental cells and resistant clones were treated with cycloheximide (100 μg/ml). Lysates were collected at the indicated times, and BRCA1, MCL-1, and tubulin protein levels were measured by Western blot. To detect BRCA1 in MDA-MB-436 parental cells, we loaded two times the amount of protein and increased film exposure time compared with RR clones. (D) MDA-MB-436 parental cells were treated with the indicated concentrations of MG132 or bortezomib (Velcade) for 24 h, and BRCA1 protein levels were measured by Western blot. 4of8

5 Fig. S5. (A) MDA-MB-436 cells expressing ectopic WT BRCA1 (MDA-MB-436+WT) were treated with vehicle (marked as V ) or 50 nm 17-DMAG in the presence of DMSO or 100 nm rucaparib, and colony formation was assessed (n = 3, mean ± SEM of colonies formed relative to vehicle + DMSO-treated cells). Because WT BRCA1 protein levels were not reduced after 17-DMAG treatment in MDA-MB-436+WT cells (Fig. 3C), we further investigated the mechanism by which 17-DMAG sensitizes to rucaparib treatment. (B) MDA-MB-436+WT cells were treated with 17-DMAG for the indicated number of hours and lysates were collected. BRCA1, BRCA2, RAD51, and tubulin levels were measured by Western blot. BRCA2 and RAD51 have previously been shown to be HSP90 client proteins. The reduction in BRCA2 and RAD51 protein levels were likely to account for the PARP inhibitor sensitization afforded by 17-DMAG in MDA-MB-436+ WT cells. Fig. S6. (A) BRCA1 was immunoprecipitated from MDA-MB-436+WT cells or resistant clones RR-1, RR-5, and RR-6, and BRCA1, BARD1, PALB2, BRCA2, RAD51, CtIP, and RAP80 protein levels were measured by Western blot (WCE, whole cell extract). (B)(Left) Western blots of protein knockdown in MCF7 and MDA-MB- 436+WT cells and resistant clones RR-1 and RR-5 using a scrambled (Sc) control or three individual sirnas targeting BRCA1 or CtIP. (Right) Detection of RPA32 and DAPI by immunofluorescence in MCF7 and MDA-MB-436+WT cells and in resistant clones RR-1 and RR-5 treated with the indicated sirnas and fixed 6 h after γ-irradiation. Representative cells are shown. (C) Quantification of foci positive cells (n = 3, mean ± SEM percentage of cells containing more than five foci). 5of8

6 Fig. S7. (A) DNA was extracted and the TP53BP1 gene was sequenced as for Fig. S2A; representative electropherograms surrounding exon 18 of the TP53BP1 gene are depicted. (B) DNA sequence surrounding exon 18 of the human TP53BP1 gene on chromosome 15 is shown. Red letters represent the region of DNA deleted in MDA-MB-436 RR clones by microhomology-mediated deletion. Yellow highlights regions of microhomology. (C) 53BP1, tubulin, and histone H3 levels were measured in cytoplasmic (marked as c ) and nuclear (marked as n ) extracts from MCF7 cells, MDA-MB-436 parental cells, and resistant clones RR- 1 to RR-6 by Western blot. Light and dark exposures of the 53BP1 blot are shown. Although the antibody used to detect 53BP1 recognizes the center of the WT 53BP1 protein, a region retained by the truncated protein, we could not detect the presence of a truncated band arising from the mutant allele. (D) MDA-MB- 436 parental cells were treated with scrambled or 53BP1 sirna, exposed to rucaparib for 72 h, replated, and assessed for colony formation after 2 wk. Colony formation was calculated as in Fig. 1A. (Inset) Western blot demonstrating 53BP1 knockdown. (E) MDA-MB-436 parental cells with stable control (+GFP) or WT BRCA1 (+BRCA1) add-backs were treated with rucaparib, and colony formation was assessed as for Fig. 1A. (Inset) Western blot demonstrating WT BRCA1 reexpression. (F) SUM1315 cells were treated with scrambled or 53BP1 sirna, exposed to rucaparib for 72 h, replated, and assessed for colony formation after 2 wk. Colony formation was calculated as in Fig. 1A. (Inset) Western blot demonstrating 53BP1 knockdown. (G) HCC1395 cells were treated with scrambled or 53BP1 sirna, exposed to rucaparib for 72 h, replated, and assessed for colony formation after 2 wk. Colony formation was calculated as in Fig. 1A. (Inset) Western blot demonstrating 53BP1 knockdown. 6of8

7 Fig. S8. (A) Detection of RPA32, RAD51, and DAPI by immunofluorescence. MDA-MB-436 RR-1 cells were infected with lentivirus containing 53BP1 cdna (53BP1) or GFP control. Cells were fixed 6 h after γ-irradiation (n = 3, mean ± SEM percentage of cells containing more than five foci). (Left) Representative cells. (Inset) 53BP1 protein levels measured by Western blot. (B) MDA-MB-436 RR-1 control (+GFP) cells or cells expressing ectopic 53BP1 were treated with rucaparib at the indicated concentrations for 2 wk, and colony formation was assessed and calculated as in Fig. 1A. (C) MDA-MB-436 parental and resistant clones were assessed for RNF8, RNF168, and tubulin protein levels by Western blot. (D) Detection of RPA32, RAD51, and DAPI by immunofluorescence. MCF7 cells were treated with scrambled (Sc) or 53BP1 sirna and fixed 6 h after γ-irradiation (n = 3, mean ± SEM percentage of cells containing more than five foci). Representative cells are shown. (Inset) Western blot demonstrating 53BP1 knockdown. (E) Quantification of percentage of cells containing more than five foci. 7of8

8 Table S1. Patient no. Characterization of BRCA1 mutant primary and recurrent ovarian carcinomas before and after platinum treatment Germ-line mutation Specimen Clinical platinum sensitivity Secondary genetic change Increase in BRCA1 staining Decrease in 53BP1 staining delAG Preplatinum Sensitive Postplatinum Resistant Yes No No delAG Preplatinum Sensitive Postplatinum Resistant Yes No No delAG Preplatinum Sensitive Postplatinum Sensitive No No No delGT Preplatinum Sensitive Postplatinum Sensitive No No No delAG Preplatinum Sensitive Postplatinum Resistant No No No delAG Preplatinum Sensitive Postplatinum Sensitive No No No 5374DON 300T>G Preplatinum Sensitive Postplatinum Sensitive Yes No No delC Preplatinum Resistant Postplatinum Resistant Yes Yes Yes C>G Preplatinum Resistant Postplatinum Resistant No Yes No delAA Preplatinum Sensitive Postplatinum Resistant No No No G>A Preplatinum Sensitive Postplatinum Resistant No No Yes C>T Preplatinum Resistant Postplatinum Resistant Yes No Yes insC Preplatinum Sensitive Postplatinum Resistant Yes No No insC Preplatinum Sensitive Postplatinum Sensitive No Yes No C>T Preplatinum Sensitive Postplatinum Resistant No Yes Yes C>T Preplatinum Sensitive Postplatinum Resistant No No No Tumors harboring BRCA1 BRCT domain mutations are shaded. 8of8