Supporting Information

Baratta et al. 10.1073/pnas.1422165112 SI Materials and Methods The genomic region encoding each relevant shRNA was am- Pooled Negative Selection shRNA In-Tumor Screen. This approach plified by PCR from genomic DNA (compare above) using two was based on the in vivo tumor screening methodology of Lowe rounds of PCR, as previously described (4). Briefly, primary PCR and Hemann (1, 2). The arrayed shRNA lentiviral TRC (The reactions were performed with Takara exTaq on up to 10 μgof RNAi Consortium) library directed at the human kinome and genomic DNA using the primary PCR primer mix (forward: ∼300 additional oncoproteins (∼8k shRNA) was described else- aatggactatcatatgcttaccgtaacttgaaagtatttcg; reverse: ctttagtttgtat- where (3). Equivalent amounts of virus from the arrayed TRC gtctgttgctattatgtctactattctttccc). The secondary PCR amplifica- shRNA library were pooled together to generate four pools (1–4) tion was performed as previously described (5), using modified of ∼2k shRNA each. Each pool was complemented with equiv- forward primers, which incorporated Illumina adapters and six- alent amounts of a set of ∼100 negative control shRNAs (CTRL nucleotide barcodes (forward universal PCR primer: 5′caagca- shRNAs) directed at the LacZ, GFP, RFP, and Luciferase . gaagacggcatacgagctcttccgatcttgtggatgaatactgccatttgtctc-3′; reverse ′ The viral titer for each pool was then determined in OVCAR8. 6nt bar-coded PCR primer: 5 aatgatacggcgaccaccgaccgtaacttg- All xenograft studies were conducted under the guidelines aaagtatttcgatttcttggctttatatatcNNNNNNaaaggac-3′). Secondary of the Dana-Farber Cancer Institute (DFCI) Animal Research PCR reactions were quantified based on their relative EtBr- Facility using approved protocols. For each lentiviral shRNA stained agarose gel band intensity. Equivalent amounts of the library pool, OVCAR8 cells were infected at a low multiplicity of secondary PCR products were pooled together and gel purified. infection (MOI = 0.3) to introduce, on average, one virus copy Samples were then sequenced using a custom sequencing primer per cell and selected by a 72-h incubation of the infected culture (CCGTAACTTGAAAGT/i6diPr/TTTCGATTTCTTGGCTTT/ with 1.5 μg/mL Puromycin. All viruses carried a PuroR . i6diPr/T/i6diPr/TATC) in an Illumina Hi-Seq machine. Aliquots of infected cells were snap frozen in ATL buffer Raw sequencing data from each sample were aligned to the (QIAGEN) and saved as reference samples, whereas the residual shRNA library barcodes, and the absolute number of reads was cells were injected either subcutaneously (SQ) or into the peri- converted into relative abundance values (number of sequence toneal cavity (IP) of immunocompromised NGS mice. reads/total reads) and normalized, within each sample, based on For SQ xenografts, 4 × 106 cells representing each shRNA the average of the reference CTRL shRNA abundance values. library pool (∼2k) were injected into each of 10 mice and al- Normalized relative abundance values thus obtained were then lowed to grow as tumors until they reached 1 cm in their greatest averaged among replicates. For each library pool, shRNAs with an μ < dimension. At this time (∼2 mo), each tumor was harvested, averaged normalized relative abundance ( i)of 0.8 in the ref- fragmented manually, and snap-frozen in ATL buffer (QIAGEN) erence sample were considered underrepresented and removed ι for further processing. from the analysis. For each shRNA iota ( ), the fold change (FC) For IP xenografts, 1 × 107 cells representing each shRNA li- value was calculated as the ratio of the averaged normalized rel- brary pool (∼2k) were injected into each of 25 mice and allowed ative abundance obtained in the tumor sample to that obtained in the reference sample (μTι/μRι). The FC z-score for each shRNA ι to grow until mice were visibly ill (about 1.5 mo). At that point, μ tumor cell-containing ascitic fluid (present in 14 of 25 mice) and was calculated in each pool relative to the average ( FC_CTRL)and standard deviation (σFC_CTRL) of the CTRL shRNA FC distri- a pool of every solid tumor mass that was grossly visible in the = − μ σ peritoneal cavity were separately collected from each mouse. bution [shRNAi FC score (FCi FC_CTRL)/ FC_CTRL]. Manually fragmented solid tumor material and ascites were then Identification of Candidate Target Genes. To account for shRNA snap- frozen in ATL buffer (QIAGEN) for further processing. off-target effects, each gene screened in the library was assigned DNA from reference and tumors samples was extracted, in an average FC value (avFC) representing the average of the FC parallel, at the end of each experiment. Fragmented subcutaneous values of all shRNAs targeting that gene. We calculated for each tumor material, originally snap-frozen in 3 mL (about 5× volume gene ι the avFC z-scores [(AvFC -μ )/σ ], and of the tumor) of ATL buffer (QIAGEN), was thawed. Two i AvFC_CTRL AvFC_CTRL only genes with an AvFC z-score <−3 were considered further. hundred microliters of 20 mg/mL Proteinase K (QIAGEN) was To increase the statistical stringency of the analysis, among the added, and samples were incubated for a minimum of 5 h at genes identified using the AvFC parameter, only those associ- 56 °C or until the material appeared to be almost homogeneous. μ ated with at least three individual targeting shRNAs, each At the end of the incubation, 40 L of 100 mg/mL RNAseA with an FC z-score <−2 were considered candidate target genes − (QIAGEN) was added, and samples were incubated for 2 min at (P < 1 × 10 5). room temperature. After addition of 3.6 mL of buffer AL (QIAGEN), samples were incubated for 10 min at 70 °C, with Functional Enrichment Analysis in Candidate Gene Pools. Candidate frequent mechanical mixing until the liquid suspension was fully target gene functional systems enrichment was queried using homogeneous. Then 3 mL of ethanol was added to each sample. STRING v9.1 (6). The biological process (GOBP) Reference samples, ascites, and IP solid tumor material were term enrichment in the set of candidate target genes was evaluated processed in a similar manner. The only difference was that the using default parameters and using the entire set of tested genes as reagent amounts were, where necessary, proportionally scaled up reference background. All GOBP terms with a P < 0.001 and false or down to accommodate differences in the starting volume of discovery rate (FDR) < 0.3 were considered. The identified and material. Reference samples were originally snap-frozen in 1 mL enriched GOBP terms were clustered using REVIGO (7) to depict of ATL buffer, IP solid tumors in 15 mL of ATL buffer, and a representative subset of GOBP categories. ascites in 3 mL of ATL buffer. DNA was then purified from an aliquot of each extract using BRD4 Genomic Analysis. GISTIC2.0 analysis was performed across the DNA Blood mini Kit (QIAGEN) according to the manu- 559 primary ovarian tumors (TCGA) as outlined previously (8, 9). facturer’s instructions. The purified DNA was concentrated by Matched mRNA expression results obtained from 316 primary EtOH/NaCl precipitation to ∼500 ng/μL for deep sequencing. ovarian tumors were obtained from the cBioPortal for Cancer

Baratta et al. www.pnas.org/cgi/content/short/1422165112 1of12 Genomics (10) to correlate mRNA expression levels with copy Lentivirus Titration. shRNA- PuroR Lentivirus titration was per- number profiles. formed in OVCAR8 cells by colony-forming assay according to established protocols. Briefly 4 × 105 OVCAR8 cells were plated Ovarian Cancer Primary Strains (DFs) and PDX Models. The ovarian in 60-mm dishes on day 0 and infected on day 1 with serial di- cancer primary strains (DFs) studied here were described pre- lutions of each lentiviral mix (with dilutions of the virus mix − − viously (11). DFs were established by implanting nude mice in- ranging from 10 4 to 10 7). Infected cells were selected with traperitoneally with fresh, primary human ovarian cancer cells Puromycin for ∼2 wk, and colonies were counted to establish the ’ purified from patients samples under a DFCI institutional re- viral titer. For the expression of lentivirus stocks that lacked the view board-approved protocol. Consistent with the correspond- PuroR cassette, titration was performed by FACS counting of ing clinical status of the donor patients, most orthotopic DF mCherry positive cells 48h after infection. models exhibited diffuse peritoneal disease with infiltration of the omentum, ovaries, pancreas, and spleen, along with gross qRT-PCR. RNA was extracted with RNAeasy Plus Kit (QIAGEN) ascites. Fresh ascites-derived DFs were collected from mice and and cDNA was generated using the SuperScript III First-Strand passaged both as nonadherent cultures and in ongoing mouse Synthesis Kit (Invitrogen) following the manufacturer’s instructions. xenograft models. To generate suitable PDX models, DFs pas- qRT-PCR reactions were performed in 384-well format with saged in vivo were infected ex vivo by mCherry-Luciferase en- SYBR-Green Supermix (Bio-Rad) using the following primer pairs: coding lentivirus to allow for noninvasive, semiquantitative BLI of tumor burden. In vivo passaged, luciferase-expressing DF cells BRD4: Fwd 5′ctccagtgagtccagctcctctgaca3′, Rev 5′gggtgcccc- ′ were further expanded and used for both ex vivo analyses and ttcttttttgacttcggagccatct3 drug efficacy studies. BUB1B: Fwd 5′tttcactccatatgtggaagagactgcac3′, Rev 5′tggtgc- ttaggatgtggtttatactaggttca3′ PDX JQ1 Efficacy Studies. All PDX studies were conducted under the guidelines of the DFCI Animal Research Facility with ap- CDK2: Fwd 5′gagtccctgttcgtacttacacccatga3′, Rev 5′gaaaga- proved protocols. Aliquots of 5 × 106 ascites-derived early pas- tccggaagagctggtcaatct3′ sage DF cells were implanted in 25 female NSG mice. At the end TRRAP: Fwd 5′gccgaattttatgcactgaagggaatg3′, Rev 5′aggcttt- of week 1, postimplant mice were subjected to BLI imaging and caccagcacatcgtgcatct3′ were randomized into two groups (10 mice each) based on mean ′ ′ ′ BLI values. The mice in each group were treated with JQ1 (50 N4BP2: Fwd 5 gcagctctttaagatatttccagccattaacc3 , Rev 5 ccctt- ′ mpk; IP route) or vehicle, as previously described (12), for 21 caagaacacagttaagaaattgcac3 (DF14-Luc and DF181-Luc) or 35 d (DF86-Luc) and monitored PRKDC: Fwd 5′tctggatcttgctgtattggagctcat3′, Rev 5′ctgaagct- for tumor burden by weekly BLI imaging until the end of the ctggtctaacatgccgttc3′ study. Body weight measurements and gross health checks were ′ ′ ′ performed twice weekly. Ascites-derived tumor samples were MAPKAPK5: Fwd 5 agtatgtcggaggagagcgacatggac3 , Rev 5 cactaattccagctcccagcttctgag3′ collected at the end of each study and snap frozen. CDC2L1: Fwd 5′ggcatcagagcagtctgccgaagaagt3′, Rev 5′ggtc- Plasmids. For validation of individual shRNAs, specific oligonu- gaaccgtgactctggaacaaccaa3′ cleotides (shRNAs clones are reported in Table S1 and Fig. 3 and PIK3CD: Fwd 5′cggagggggctttgctggtctttcttg3′, Rev 5′cacccca- their sequences are publicly available through the TRC portal ′ www.broadinstitute.org/rnai/public/) were annealed and cloned, gggggcatcctgcgttgtta3 as described by TRC published protocols, into the pLKO.1 de- TYRO3: Fwd 5′ccaggtgctgatggccgagctctgct3′, Rev 5′acttccca- rivative lentiviral vector TRC047 (pLKO.3pgw). This plasmid gcctcctggggcctgtgt3′ contains a GFP/PuroR cassette encoding a polycistronic PuroR- ATM: Fwd 5′caggcagaaaaagatgcaggaaatcagtag3′, Rev 5′taaga- F2A-eGFP cDNA cloned in place of the PuroR cassette. For gttcttgacattttagcctaggtgct3′ BRD4 rescue experiments, the wild-type human BRD4 long isoform cDNA (NM_058243, from Addgene plasmid 22304) (13) STK3: Fwd 5′acagcaacgagaattggaagaggaaga3′, Rev 5′ccacact- was cloned into a pLVX-puro (Clontech) derivative lentiviral ctccacactagtcttcacca3′ vector from which the PuroR cassette was deleted. BRD4 was CALM2: Fwd 5′tgaccaactgactgaagagcagattgcag3′, Rev 5′ccc- expressed as a mCherry-T2A-bsr-P2A-hBRD4 polycistronic aattcctttgttgttatagttccatcaccatc3′ cDNA (pLVX-Cherry-Brd4). The intervening T2A and P2A self- cleaving peptides (14) result in translation of mCherry, blastici- PRKAA1: Fwd 5′gggtcggcaccttcggcaaagtgaag3′, Rev 5′agg- din resistance gene (bsr,) and hBRD4 as discrete polypeptides ctccgaatcttctgtcgattgagt3′ encoded by a single mRNA. The control expression vector, PFKFB3: Fwd 5′gagtcggtgtgcgacgaccctacagtt3′, Rev 5′cagtcttt- pLVX-Cherry, lacks BRD4 cDNA. gtaatccgggctggagat3′

Cell Culture. The OVCAR8 cells used in this study stably express c-MYC (15): Fwd 5′agggatcgcgctgagtataa3′, Rev 5′tgcctctcgct- the Luciferase gene (11) and were cultured in RPMI media ggaattact3′ supplemented with 10% FBS (Gibco) and Pen-Strep (Gibco). MYCN (16): Fwd 5′ctcagtacctccggagag3′,Rev5′ggcatcgttt- The DFs were cultured as nonadherent cultures in RPMI media gaggatc3′ supplemented with 10% FBS (Gibco) and antibiotic-antimycotic ′ ′ ′ (Gibco), which was replaced by Pen-Strep for virus infections. 36B4: Fwd 5 atcaacgggtacaaacgagtcctg3 , Rev 5 aaggcagatgga- ′ OVCAR8 and primary strain (DFs) infections were performed tcagccaagaag3 by incubating cells for 24 h with the indicated amount of lenti- TUBULIN: Fwd 5′gggaaatcgtgcacatcca3′,Rev5′ccaggatggc- virus in the presence of 8 μg /mL polybrene. Where described, acgaggaa3′ infected cells were selected in the presence of 1.5 μg/mL Puro- mycin. All lentiviral packaging was performed by transfecting Cell Extracts and Western Blotting. Extracts were prepared by lysing 293T cells according to established protocols using pCMV- cells in 10 volumes (with respect to pellet size) of RIPA buffer ΔR8.91 and pCMV-VSV-G as packaging plasmids. supplemented with 1 mM PMSF, 50 mM NaF, 1 mM NaVO4, and

Baratta et al. www.pnas.org/cgi/content/short/1422165112 2of12 1× protease inhibitor mixture solution (Santa Cruz Biotech- coinfection with shLuc (a control shRNA not affecting pro- nology). Extracts were resolved by SDS gel electrophoresis and liferation), shPLK1 (a control shRNA affecting proliferation in- blotted onto nitrocellulose membranes. The following antibodies dependently of BRD4), or three different shBRD4s (1, 2, and 3). were used to probe Western blots: anti-BRD4 Ab (Bethyl A301- In each infection, the GFP- and the Cherry-positive cell per- 985A and Abcam 75898), anti-cMYC Ab (c-Myc (9E10):sc-40 centages were assessed by FACS in aliquots of infected cells at and c-Myc (N-262):sc-764; Santa Cruz Biotechnology), anti- 48 h after infection (day 0) and at subsequent time points as MYCN Ab (N-Myc (C-19):sc-791; Santa Cruz Biotechnology), indicated in the text. The GFP- and Cherry-positive cell per- and anti-actin (A2066; Sigma-Aldrich), used as a loading control. centage at each time point was then expressed as percentage of the value obtained at day 0. Flow Cytometry (FACS) Analysis and Sorting. For FACS analysis, infected cells were trypsinized and fixed in a 3% parafor- BRD4 Inhibitor Dose–Response Curves. OVCAR8 cells were plated maldehyde solution, washed once in PBS, and analyzed by flow in 96-well plates (5 × 103 cells per well). Where shRNAs were cytometry for GFP and/or mCherry signals. For FACS, infected tested, OVCAR8 cells were infected with the appropriate cells were trypsinized, resuspended in PBS, and sorted for their shRNA-encoding lentivirus and selected for 72 h in 1.5 μg/mL GFP and/or mCherry signal. Puromycin before seeding on 96-well plates. DF cells were plated in polyhema-treated 96-well plates at an input density of OVCAR8/shRNA Proliferation Assay. OVCAR8 cells were plated in 0.25 μL pellet volume/100 μL in each well. We tested the effect 96-well plates (5 × 103 cells per well) and infected with shRNA- ∼ of three different small molecules reported to inhibit BRD4 expressing lentivirus at MOI 10. Cells were selected for 48 h in activity: JQ1 (12), I-BET-762 (17), and I-BET-151 (18). For each Puromycin. A luciferase assay (Promega) was performed im- BRD4 inhibitor, cells were exposed to 1:2 serial dilutions of the mediately after Puromycin selection (day 0) and 4 d later to compound over a concentration range of 5 μMto∼5 nM and monitor cell proliferation. incubated for 48 h. Then, the drug treatment was repeated, and GFP Competition Assays. OVCAR8 and primary ovarian cancer cells were incubated for an additional 48 h at 37 °C. Cell viability cells were infected with each of the shRNA/GFP-expressing was assessed at 96 h by Cell Titer Glo (Promega). Results from lentiviruses at an MOI between 0.1 and 0.2. The population each data point were reported as a percentage of the signal percentage of GFP-positive cells was then followed over time to generated by drug-treated relative to vehicle-treated cells for – establish their growth rate with respect to the GFP-negative each cell type. Dose response curves were generated using population. GraphPad Prism 6.0. OVCAR8 cells were plated in six-well plates (2 × 105 cells per μ JQ1 ex Vivo Proliferation Curves. OVCAR8 cells were plated in 8 × well) and infected with 10 L of each viral suspension. DFs were × 3 infected with 3.5 mL of the same viral suspension to achieve 96-well plates (5 10 cells per well) and treated at day 0 with a similar percentage of infected cells. In each infection, the the indicated amount of JQ1-S or JQ1-R. Each plate was assayed percentage of GFP-positive cells was assessed by FACS in ali- by Cell Titer Glow (Promega) at day 0 and every 2 d thereafter ∼ quots of infected cells at day 0 (48 h after infection for OVCAR8 for 2 wk. and 96 h for DFs) and at subsequent time points, as indicated in For DF cell analysis, the DF pellet size was determined, after the text. The GFP-positive cell percentage at each time point was which that DF was plated on polyhema-treated, i-well plates at μ then expressed as percentage of the value obtained at day 0. a density of 10 L pellet volume/2 mL of medium in each well. Each well culture was treated at day 0 with the indicated amount BRD4 Rescue Experiment. OVCAR8 cells were plated in six-well of JQ1-S or JQ1-R. Aliquots of 100 μL (5% total volume) from plates (2 × 105 cells per well) and infected at MOI = 0.3 with either each well were assayed by Cell Titer Glo at day 0 and every 48 h pLVX-Cherry-BRD4 or pLVX-Cherry expression vectors. Im- thereafter for ∼2 wk. JQ1 treatment for OVCAR8 and DF mediately following this first infection, individual shRNA/GFP strains was repeated every 48 h. Results of each treatment were lentivirus mixes were added to achieve a total MOI = 0.6. For reported relative to the metabolic activity observed at day 0. each pLVX-Cherry vector (BRD4 containing or empty), we tested Growth curves were generated using GraphPad Prism 6.0.

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Baratta et al. www.pnas.org/cgi/content/short/1422165112 3of12 Fig. S1. Candidate target gene evaluation in two ovarian cancer primary strains DF14 and DF37. (A) qRT-PCR analysis of candidate genes expression in DF14 and DF37. For each gene, expression values are normalized by reference to that of the housekeeping gene, 36B4, and are reported as a percentage of the levels obtained in OVCAR8 (OV8). (B and C) GFP competition assay results in DF14 (B) and DF37 (C).

Baratta et al. www.pnas.org/cgi/content/short/1422165112 4of12 Fig. S2. Ectopic BRD4 expression rescues the effect of shBRD4 in OVCAR8 (OV8). (A) qRT-PCR analysis of BRD4 expression in OV8 after infection at MOI > 1and 48-h selection in puromycin with the indicated shRNAs. Expression levels are normalized to tubulin expression and reported as a percentage of the value obtained on infection with the negative control hairpin shLUC. The negative control hairpin shCTRL is also shown. (B) BRD4 reconstitution experiment outline. OV8 cells were infected at total MOI = 0.6 with equivalent amounts of the Cherry-BRD4 lentivirus and a GFP/shRNA lentivirus to obtain four different target cell populations to follow by FACS over time: GFP+ (expressing only the shRNA); Cherry+ (expressing only BRD4); GFP+/Cherry+ (expressing both the shRNA and BRD4); DN (double negative GFP−/Cherry− expressing none of the ectopic DNA). Reported is a representative FACS result of all coinfections at 48 h after infection. Each coinfection was performed with a single shRNA/GFP carrying lentivirus: shLuc (negative control), shPLK1 (positive control), and shBRD4 1, 2, and 3. (C)GFP+ population evolution in time (these cells express only the indicated shRNA). (D)GFP+/Cherry+ population (these cells express ectopic BRD4 plus the indicated shRNA). (E–G) Same experiment described above but coinfections were carried out by using the empty Cherry-expressing lentiviral vector.

Baratta et al. www.pnas.org/cgi/content/short/1422165112 5of12 Fig. S3. DFs Sensitivity to BRD4 inhibition. (A) JQ1 sensitivity across DF strains. In the table, for each DF, are reported the Emax values obtained from each JQ1 dose– response curve. For each value, 95% CI is shown. DF sensitivity is ranked according to the JQ1 Emax value into five groups. (B and C) Comparison of dose–response curves obtained with three different BRD4 inhibitors (JQ1, I-BET-762, and I-BET-151) in two JQ1 highly sensitive DFs (B) and in two JQ1-refractory DFs (C).

Fig. S4. BRD4 depletion impairs proliferation of DF14 and DF37. (A and B) GFP competition assay in DF14 (A)orDF37(B) using shLuc (negative control shRNA), shPLK1 (positive control shRNA), or three different shBRD4 hairpins (1, 2, and 3). Here is reported for each shRNA the percentage of the GFP population that is present at each indicated time point, relative to the value obtained 4 d after infection (day 0). (C) Western blot analysis of BRD4 expression of DF14 cells sorted by FACS on the basis of GFP expression. (D) Western blot analysis of BRD4 and N-Myc expression of DF14 and DF37 cells sorted by FACS on the basis of GFP expression at day 7. Actin was used as a loading control.

Baratta et al. www.pnas.org/cgi/content/short/1422165112 6of12 Fig. S5. Proliferation curves of DF14 and DF37 at different JQ1 and JQ1-R concentrations. DF14 (A) and DF37 (B) proliferation curves in the presence of different concentration of JQ1 (JQ1-S). DF14 (C) and DF37 (D) proliferation curves in the presence of different concentration of the JQ1 inactive enantiomer JQ1-R. Cell viability is measured by titering ATP levels with the Cell Titer Glow assay and is reported as a percentage of the levels obtained on day 0.

Baratta et al. www.pnas.org/cgi/content/short/1422165112 7of12 Fig. S6. BRD4 is amplified in ovarian cancer. (A) GISTIC2.0 analysis across 559 SNP6.0 Affymetrix copy number arrays from primary ovarian tumors (TCGA) reveals a significant amplification peak in a region containing the BRD4 gene (Left). Heat map depicting relative copy number of 559 primary ovarian cancers reveals high-level amplification of the region containing BRD4 in 19% of all cancers (Center). The amplification peak with BRD4 also includes EPHX3, whereas NOTCH3 is adjacent to the amplification peak (Right). (B) Analysis of mRNA expression and copy number of BRD4 in 316 primary ovarian cancers (TCGA) reveals a correlation between mRNA expression and copy number of BRD4.

Fig. S7. Anticorrelation between c-MYC and MYCN expression in ovarian cancer samples and c-MYC/MYCN down-regulation in DFs following BRD4 inhibition. (A) Pearson correlation analysis of c-MYC and MYCN expression in ovarian cancer tumor samples downloaded from the cBioportal TCGA public database. (B and C) Western blot showing MYCN (B) and c-MYC (C) down-regulation in JQ1-sensitive DFs (highlighted in red) following 48-h treatment with three different BRD4 inhibitors (JQ1, I-BET-762, and I-BET-151). JQ1-refractory strains (DF181 and DF164, highlighted in blue) are also shown.

Baratta et al. www.pnas.org/cgi/content/short/1422165112 8of12 aat tal. et Baratta Table S1. Candidate target genes list No. shRNA Best shRNA Second best shRNA Third best shRNA Library tested in Average Gene name pool the screen TRC clone z-score TRC clone z-score TRC clone z-score FC Gene description Protein ID www.pnas.org/cgi/content/short/1422165112 ERBB3 1 5 TRCN0000000619 −3.33 TRCN0000000621 −3.19 TRCN0000000622 −2.95 0.52 v-erb-b2 erythroblastic leukemia viral P21860 oncogene homolog 3 (avian) PRKAA1 1 5 TRCN0000000861 −3.33 TRCN0000000858 −2.80 TRCN0000000859 −2.42 0.44 Protein , AMP-activated, alpha Q13131 1catalytic subunit BUB1B 1 10 TRCN0000194865 −3.33 TRCN0000000464 −3.30 TRCN0000000465 −3.30 0.22 Budding uninhibited by benzimidazoles 1 O60566 homolog beta (yeast) CDC2L1(CDK11B) 1 9 TRCN0000006209 −3.29 TRCN0000196705 −3.05 TRCN0000006207 −3.00 0.40 Cyclin-dependent kinase 11B P21127 PRKDC 1 10 TRCN0000196328 −3.10 TRCN0000006256 −2.58 TRCN0000006257 −2.43 0.60 Protein kinase, DNA-activated, catalytic P78527 polypeptide BLK 1 9 TRCN0000195414 −3.09 TRCN0000010086 −3.03 TRCN0000197266 −2.63 0.59 B lymphoid tyrosine kinase P51451 FRAP1(mTOR) 1 10 TRCN0000039784 −3.06 TRCN0000039783 −2.99 TRCN0000039785 −2.94 0.52 Mechanistic target of rapamycin (serine/ P42345 threonine kinase) PI4KAP2 3–4 15 TRCN0000078691 −3.05 TRCN0000078690 −2.85 TRCN0000052623 −2.62 0.44 Phosphatidylinositol 4-kinase, catalytic, alpha pseudogene 2 GALK2 1 8 TRCN0000010099 −3.04 TRCN0000010092 −2.64 TRCN0000010100 −2.59 0.54 Galactokinase 2 Q01415 AURKA 3 9 TRCN0000010533 −3.04 TRCN0000196732 −2.95 TRCN0000000658 −2.81 0.47 A O14965 CDK2 3 9 TRCN0000000587 −3.03 TRCN0000010470 −2.60 TRCN0000039959 −2.31 0.60 Cyclin-dependent kinase 2 P24941 CKB 3 9 TRCN0000194869 −3.00 TRCN0000010991 −2.73 TRCN0000199638 −2.50 0.59 Creatine kinase, brain P12277 3 8 TRCN0000121222 −2.98 TRCN0000121075 −2.91 TRCN0000121072 −2.43 0.51 Polo-like kinase 1 (Drosophila) P53350 NME1 (NM23A) 3 6 TRCN0000010064 −2.92 TRCN0000197006 −2.46 TRCN0000199648 −2.23 0.57 Nonmetastatic cells 1, protein (NM23A) P15531 GLYCTK 3 5 TRCN0000016932 −2.90 TRCN0000016929 −2.85 TRCN0000016931 −2.58 0.32 Glycerate kinase Q8IVS8 FASTKD3 3 9 TRCN0000148034 −2.82 TRCN0000127765 −2.74 TRCN0000147580 −2.58 0.61 FAST kinase domains 3 Q14CZ7 RPS6KA4 4 10 TRCN0000021515 −2.77 TRCN0000021514 −2.57 TRCN0000021516 −2.45 0.55 Ribosomal protein S6 kinase, 90kDa, O75676 polypeptide 4 TEC 4 7 TRCN0000009992 −2.73 TRCN0000195027 −2.37 TRCN0000009983 −2.05 0.57 tec protein tyrosine kinase P42680 PRKAB2 4 8 TRCN0000003134 −2.70 TRCN0000003132 −2.34 TRCN0000003131 −2.25 0.61 Protein kinase, AMP-activated, beta 2 O43741 noncatalytic subunit MAPKAPK5 2 9 TRCN0000195129 −2.66 TRCN0000000681 −2.59 TRCN0000000683 −2.58 0.48 Mitogen-activated protein kinase-activated Q8IW41 protein kinase 5 VRK1 2 9 TRCN0000002133 −2.66 TRCN0000199332 −2.61 TRCN0000197134 −2.56 0.43 Vaccinia related kinase 1 Q99986 BRD4 2 10 TRCN0000021427 −2.66 TRCN0000196576 −2.24 TRCN0000021428 −2.01 0.57 Bromodomain containing 4 O60885 IHPK2 (IP6K2) 3 8 TRCN0000199807 −2.65 TRCN0000199458 −2.41 TRCN0000195257 −2.10 0.55 Inositol hexakisphosphate kinase 2 Q9UHH9 TRRAP 4 5 TRCN0000005364 −2.63 TRCN0000005361 −2.62 TRCN0000005365 −2.24 0.34 Transformation/transcription domain- Q9Y4A5 associated protein ZAP70 2 8 TRCN0000199795 −2.62 TRCN0000000438 −2.33 TRCN0000199778 −2.10 0.57 Zeta-chain (TCR) associated protein kinase P43403 70 kDa BCR 2 9 TRCN0000195608 −2.61 TRCN0000194953 −2.48 TRCN0000000791 −2.29 0.53 Breakpoint cluster region P11274 RIPK5 2 10 TRCN0000196529 −2.59 TRCN0000037509 −2.30 TRCN0000195331 −2.11 0.54 Dual serine/threonine and tyrosine protein Q6XUX3 kinase ATM 2 9 TRCN0000038656 −2.58 TRCN0000038654 −2.40 TRCN0000038658 −2.09 0.47 Serine-protein kinase ATM (Ataxia Q13315 telangiectasia mutated) PIK3C2A 2 8 TRCN0000002230 −2.57 TRCN0000194822 −2.38 TRCN0000002232 −2.24 0.61 Phosphoinositide-3-kinase, class 2, alpha O00443 polypeptide PIK3CD 2 7 TRCN0000196921 −2.57 TRCN0000033277 −2.21 TRCN0000033278 −2.01 0.48 Phosphoinositide-3-kinase, catalytic, delta O00329 9of12 polypeptide aat tal. et Baratta Table S1. Cont. No. shRNA Best shRNA Second best shRNA Third best shRNA Library tested in Average

www.pnas.org/cgi/content/short/1422165112 Gene name pool the screen TRC clone z-score TRC clone z-score TRC clone z-score FC Gene description Protein ID

RPS6KA6 2 10 TRCN0000196796 −2.57 TRCN0000002265 −2.12 TRCN0000196549 −2.05 0.62 Ribosomal protein S6 kinase, 90kDa, Q9U.K.32 polypeptide 6 TYRO3 4 8 TRCN0000002179 −2.55 TRCN0000002181 −2.46 TRCN0000195673 −2.40 0.37 TYRO3 protein tyrosine kinase Q06418 SGK269 (PEAK1) 4 10 TRCN0000195001 −2.55 TRCN0000195688 −2.43 TRCN0000037441 −2.01 0.60 NKF3 kinase family member Q9H792 RAF1 2 9 TRCN0000001068 −2.52 TRCN0000195502 −2.30 TRCN0000001066 −2.27 0.56 v-raf-1 murine leukemia viral oncogene P04049 homolog 1 PFKFB3 4 5 TRCN0000007342 −2.51 TRCN0000195650 −2.23 TRCN0000007341 −2.14 0.49 6-Phosphofructo-2-kinase/ Q16875 fructose-2,6-biphosphatase 3 SMG1 2 9 TRCN0000037410 −2.50 TRCN0000194789 −2.48 TRCN0000037409 −2.23 0.60 SMG1 homolog, phosphatidylinositol Q96Q15 3-kinase-related kinase (C.elegans) STK3 2 5 TRCN0000002176 −2.50 TRCN0000002173 −2.49 TRCN0000002175 −2.46 0.43 Serine/threonine kinase 3 (STE20 Q13188 homolog, yeast) N4BP2 2 4 TRCN0000051693 −2.48 TRCN0000051695 −2.22 TRCN0000051697 −2.02 0.22 NEDD4 binding protein 2 Q86UW6 FGFR1 4 5 TRCN0000121106 −2.43 TRCN0000121105 −2.31 TRCN0000000420 −2.25 0.48 Fibroblast growth factor receptor 1 P11362 CALM2 2 9 TRCN0000196473 −2.35 TRCN0000052580 −2.26 TRCN0000196508 −2.23 0.53 Calmodulin 2 (, delta) P62158

The candidate target genes are sorted in this table according to the z-score obtained with the best shRNA. 0o 12 of 10 Table S2. GOBP terms enriched in the pool of candidate genes Representative GOBP Number categories Term_ID Description of genes Genes P value P value_fdr

GO:0009057: Macromolecule GO:0009057 Macromolecule catabolic 7 NME1, MTOR, CDK2, AURKA, 0.00010 0.09 catabolic process process PLK1, SMG1, BUB1B GO:0019941 Modification-dependent 4 AURKA, PLK1, BUB1B, CDK2 0.00019 0.09 protein catabolic process GO:0043632 Modification-dependent 4 AURKA, PLK1, BUB1B, CDK2 0.00036 0.14 macromolecule catabolic process GO:0030163 Protein catabolic process 6 ATM, MTOR, CDK2, AURKA, 0.00000 0.04 PLK1, BUB1B GO:0044257 Cellular protein catabolic 4 AURKA, PLK1, BUB1B, CDK2 0.00036 0.14 process GO:0043161 Proteasomal 4 AURKA, PLK1, CDK2, BUB1B 0.00019 0.09 ubiquitin-dependent protein catabolic process GO:0006511 Ubiquitin-dependent 4 AURKA, PLK1, BUB1B, CDK2 0.00019 0.09 protein catabolic process GO:0044248 Cellular catabolic process 8 NME1, CDK2, ATM, AURKA, 0.00012 0.09 BUB1B, PRKAA1, PLK1, SMG1 GO:0031145 Anaphase-promoting 4 AURKA, PLK1, BUB1B, CDK2 0.00008 0.09 complex-dependent proteasomal ubiquitin-dependent protein catabolic process GO:0051603 Proteolysis involved in 4 AURKA, PLK1, BUB1B, CDK2 0.00019 0.09 cellular protein catabolic process GO:0044265 Cellular macromolecule 7 ATM, NME1, CDK2, AURKA, 0.00001 0.04 catabolic process PLK1, SMG1, BUB1B GO:0010498 Proteasomal protein 4 AURKA, PLK1, BUB1B, CDK2 0.00019 0.09 catabolic process GO:0042770: Signal transduction GO:0042770 Signal transduction in 4 ATM, RPS6KA6, CDK2, PRKDC 0.00036 0.14 in response to DNA damage response to DNA damage GO:1902400 Intracellular signal 3 PRKDC, ATM, CDK2 0.00013 0.09 transduction involved in G1 DNA damage checkpoint GO:1902402 Signal transduction 3 PRKDC, ATM, CDK2 0.00013 0.09 involved in mitotic DNA damage checkpoint GO:0044783 G1 DNA damage 3 PRKDC, ATM, CDK2 0.00048 0.17 checkpoint GO:0072401 Signal transduction 3 PRKDC, ATM, CDK2 0.00013 0.09 involved in DNA integrity checkpoint GO:0072422 Signal transduction 3 PRKDC, ATM, CDK2 0.00013 0.09 involved in DNA damage checkpoint GO:0051276: GO:0051276 Chromosome organization 8 ATM, TRRAP, CDK2, PRKDC, 0.00010 0.09 organization PRKAA1, PLK1, VRK1, RPS6KA4 GO:0016568 Chromatin modification 6 ATM, TRRAP, CDK2, PRKAA1, 0.00038 0.14 VRK1, RPS6KA4 GO:0016572 Histone phosphorylation 5 PRKAA1, RPS6KA4, ATM, 0.00026 0.11 CDK2, VRK1 GO:0016570 Histone modification 7 VRK1, ATM, RPS6KA4, TRRAP, 0.00001 0.04 BRD4, CDK2, PRKAA1 GO:0016569 Covalent chromatin 7 VRK1, ATM, RPS6KA4, TRRAP, 0.00001 0.04 modification BRD4, CDK2, PRKAA1 GO:0006325 Chromatin organization 6 ATM, TRRAP, CDK2, PRKAA1, 0.00083 0.28 VRK1, RPS6KA4 GO:0035404 Histone-serine 4 PRKAA1, VRK1, RPS6KA4, ATM 0.00008 0.09 phosphorylation

Baratta et al. www.pnas.org/cgi/content/short/1422165112 11 of 12 Table S2. Cont. Representative GOBP Number categories Term_ID Description of genes Genes P value P value_fdr

GO:0007346: Regulation of GO:0007346 Regulation of mitotic 8 AURKA, PLK1, ATM, TRRAP, 0.00088 0.28 mitotic cell cycle cell cycle BUB1B, BRD4, CDK2, PRKDC GO:1901987 Regulation of cell cycle 7 PLK1, ATM, TRRAP, BUB1B, 0.00022 0.10 phase transition BRD4, CDK2, PRKDC GO:1901988 Negative regulation of 6 PLK1, ATM, CDK2, PRKDC, 0.00014 0.09 cell cycle phase transition TRRAP, BUB1B GO:0072395 Signal transduction involved 3 PRKDC, ATM, CDK2 0.00013 0.09 in cell cycle checkpoint GO:1901990 Regulation of mitotic cell 7 PLK1, ATM, TRRAP, BUB1B, 0.00017 0.09 cycle phase transition BRD4, CDK2, PRKDC GO:1901991 Negative regulation of 6 PLK1, ATM, CDK2, PRKDC, 0.00014 0.09 mitotic cell cycle phase TRRAP, BUB1B transition GO:0072431 Signal transduction involved 3 PRKDC, ATM, CDK2 0.00013 0.09 in mitotic G1 DNA damage checkpoint GO:0031571 Mitotic G1 DNA damage 3 PRKDC, ATM, CDK2 0.00048 0.17 checkpoint GO:0010948 Negative regulation of cell 7 PLK1, ATM, TRRAP, BUB1B, 0.00022 0.10 cycle process BRD4, CDK2, PRKDC GO:0072413 Signal transduction involved 3 PRKDC, ATM, CDK2 0.00013 0.09 in mitotic cell cycle checkpoint GO:0007093 Mitotic cell cycle checkpoint 6 PLK1, ATM, CDK2, PRKDC, 0.00010 0.09 TRRAP, BUB1B GO:0051439 Regulation of 3 PLK1, BUB1B, CDK2 0.00048 0.17 ubiquitin-protein activity involved in mitotic cell cycle

Enrichment is calculated with respect to the pool of all of the genes tested in the screen as described in SI Materials and Methods.

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