Synthetic Lethality Between CCNE1 Ampli Fication and Loss of BRCA1

Synthetic lethality between CCNE1 ampliﬁcation and loss of BRCA1

Dariush Etemadmoghadama,b,c, Barbara A. Weird,e, George Au-Yeunga,f, Kathryn Alsopa,f, Gillian Mitchella,b, Joshy Georgea,f, Australian Ovarian Cancer Study Groupa,g,h,i,1, Sally Davisa,c, Alan D. D’Andread, Kaylene Simpsonb,c,j, William C. Hahnd,e, and David D. L. Bowtella,b,c,f,2

aDepartment of Research, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia; bSir Peter MacCallum Cancer Centre Department of Oncology, University of Melbourne, Melbourne, VIC 3010, Australia; cDepartment of Pathology, University of Melbourne, Melbourne, VIC 3010, Australia; dDepartment of Medical Oncology and Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; eCancer Program, The Broad Institute of Harvard and MIT, Cambridge, MA 02142; fDepartment of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, VIC 3010, Australia; gWestmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Sydney, NSW 2145, Australia; hDepartment of Gynaecological Oncology, Westmead Hospital, Sydney, NSW 2145, Australia; iCancer Program, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia; and jVictorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia

Edited by Elizabeth M. Swisher, University of Washington, Seattle, WA, and accepted by the Editorial Board October 12, 2013 (received for review July 29, 2013) High-grade serous ovarian cancers (HGSCs) are characterized by genes inactivated by deletion, mutation, or hypermethylation in- a high frequency of TP53 mutations, BRCA1/2 inactivation, homol- clude ATM, ATR, RAD51C,andPTEN (∼10%), key Fanconi ogous recombination dysfunction, and widespread copy number anemia members (∼5%), and amplification or mutation of EMSY changes. Cyclin E1 (CCNE1) gene amplification has been reported (∼8%). Collectively, at least 50% of HGSCs are thought to have to occur independently of BRCA1/2 mutation, and it is associated HR pathway defects (9). with primary treatment failure and reduced patient survival. In- Approximately 30% of HGSC tumors have alterations in the sensitivity of CCNE1-amplified tumors to platinum cross-linking Rb pathway or genes involved in Rb-mediated DNA repair and agents may be partly because of an intact BRCA1/2 pathway. Both cell cycle control, including amplification of CCNE1 (∼20%), BRCA1/2 dysfunction and CCNE1 amplification are known to pro- loss of RB1 (∼10%), or gain of RBBP8 (∼4%) (10). Strikingly, mote genomic instability and tumor progression. These events activation of the RB1/CCNE1 pathway is largely exclusive of may be mutually exclusive, because either change provides a path BRCA1/2 mutation for reasons that are unclear (9, 10). Both to tumor development, with no selective advantage to having BRCA1/2 dysfunction and CCNE1 amplification are known to both mutations. Using data from a genome-wide shRNA synthetic promote genomic instability and tumor progression (4, 11); lethal screen, we show that BRCA1 and members of the ubiquitin therefore, they may be mutually exclusive, because either change pathway are selectively required in cancers that harbor CCNE1 provides a path to tumor development, with no selective ad- amplification. Furthermore, we show specific sensitivity of CCNE1- vantage to having both mutations (10). Insensitivity of CCNE1- amplified tumor cells to the proteasome inhibitor bortezomib. amplified tumors to platinum cross-linking agents may be partly These findings provide an explanation for the observed mutual because of an intact BRCA1/2 pathway, suggesting that these exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC patients are unlikely to respond to poly-ADP-ribose polymerase and suggest a unique therapeutic approach for treatment-resistant (PARP) inhibitors. CCNE1-amplified tumors. Significance RNAi | pan-cancer | CDK2 | cell cycle | DNA repair Women with high-grade serous ovarian cancer (HGSC) har- pithelial ovarian cancer is complex and histologically diverse boring Cyclin E1 (CCNE1) gene amplification generally face Ebut still largely treated as a single disease with limited a poor clinical outcome. These tumors comprise a significant stratification based on histological or molecular characteristics. group of ∼20% of HGSCs that are not associated with BRCA1/2 High-grade serous ovarian cancer (HGSC) accounts for the mutation and are unlikely to respond to standard cytotoxic or majority of epithelial ovarian cancer-related deaths (>60%), and poly-ADP-ribose polymerase inhibitors. We identified a specific almost no improvement in survival has been observed in the last dependency on BRCA1 and members of the ubiquitin pathway 20 y (1). Widespread copy number changes are a hallmark of in CCNE1-amplified tumors. The requirement for BRCA1 seems

HGSC, including focal amplification of Cyclin E1 (encoded by to account for the mutual exclusivity of mutations observed GENETICS CCNE1), which is associated with primary treatment failure (2) in primary tumors. We propose a unique therapeutic strategy and reduced survival (3). Amplification of CCNE1 is one of very involving inhibition of the proteasome and homologous re- few well-defined molecular targets in HGSC. combination function with bortezomib. Our findings are likely Cyclin E1 forms a complex with cyclin-dependent kinase 2 to have relevance to the treatment of other tumor types with (CDK2) to regulate G1/S transition as well as having kinase-in- CCNE1 amplification, including triple negative breast cancer. dependent functions, including in DNA replication (4). Ovarian cell lines with CCNE1 amplification show a specific dependency Author contributions: D.E., B.A.W., A.D.D., W.C.H., and D.D.L.B. designed research; D.E., B.A.W., G.A.-Y., K.A., G.M., and A.O.C.S.G. performed research; S.D. and K.S. con- for maintenance of CCNE1 expression (5, 6). We have validated tributed new reagents/analytic tools; D.E., B.A.W., G.A.-Y., K.A., G.M., J.G., and D.D.L.B. CDK2 as a therapeutic target by showing selective sensitivity to analyzed data; and D.E. and D.D.L.B. wrote the paper. suppression either by gene knockdown or using small molecule The authors declare no conflict of interest. inhibitors (7), consistent with findings in breast cancer (8). This article is a PNAS Direct Submission. E.M.S. is a guest editor invited by the Recent genomic studies have revealed a high frequency of Editorial Board. BRCA1/2 (Breast cancer 1/2, early onset) inactivation and ho- Freely available online through the PNAS open access option. mologous recombination (HR) dysfunction in HGSC (9). Alter- 1A complete list of the Australian Ovarian Cancer Study Group can be found in SI Text. ations of genes in the HR pathway include germ-line and somatic 2To whom correspondence should be addressed. E-mail: [email protected]. ∼ mutations of BRCA1 or BRCA2 ( 20% of patients) and epige- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. netic silencing of BRCA1 by hypermethylation (∼10%). Other 1073/pnas.1314302110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1314302110 PNAS | November 26, 2013 | vol. 110 | no. 48 | 19489–19494 Downloaded by guest on September 30, 2021 Here, we show that BRCA1 and members of the ubiquitin Dependencies of CCNE1-Amplified Cell Lines. We have recently pathway are selectively required in cancers that harbor CCNE1 shown oncogene addiction to Cyclin E1 and its partner kinase, amplifications. Furthermore, we show specificsensitivityofCCNE1- CDK2, in CCNE1-amplified ovarian tumors (7), suggesting that amplified tumor cells to the proteasome inhibitor bortezomib. use of CDK2 inhibitors may be effective in these cancers. In These findings provide an explanation for the observed mutual addition to CDK2, Cyclin E1 interacts with CDK1 and CDK3 exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSCs and has kinase-independent functions (4). Furthermore, Cyclin E1 and suggest a unique therapeutic approach for treatment-resistant is regulated both positively and negatively by posttranslational CCNE1-amplified tumors. proteolysis (15). To better understand the dependencies of tumor cells with CCNE1 amplification and identify other potential Results therapeutic targets, we analyzed data from a genome-wide CCNE1 Gene Amplification in Primary Tumors. To better define the shRNA screen of 102 cancer cell lines with known copy number frequency of CCNE1 amplification in solid cancers, we used status, including a high proportion of epithelial ovarian cancer (n = 25) (16). We included all available cell lines to obtain suf- genomic data from The Cancer Genome Atlas (TCGA) to per- ficient statistical power for the analysis. Cells infected with a pool form a pan-cancer analysis of 22 cancer types (Materials and of 54,020 shRNAs (targeting 11,194 genes) were grown for at Methods). We found that focal high-level amplification of 19q12 ∼ least 16 doublings, and the abundance of individual shRNA involving CCNE1 occurs at a frequency of 5% in breast, lung, sequences was measured relative to a reference to identify genes ∼ and gastric cancers and that it is most frequent ( 25%) in essential for survival (16). In two separate analyses, we compared fi HGSCs (Fig. 1A). Consistent with our previous ndings (5), the CCNE1-amplified (n = 23) with nonamplified (n = 43) and most significant or peak region of amplification always involved CCNE1 high- (n = 15) with low-expressing (n = 41) cells. To CCNE1; however, genes neighboring CCNE1, such as prefoldin- improve specificity, we removed samples with CCNE1 copy like chaperone URI1, were present in peak regions in some tu- number or expression that fell into the midrange of values. mor types. These findings suggest that the reported driver ac- Using a statistical approach that considered data from either tivity of URI (12) or other genes within the 19q12 amplicon (8) the second-best shRNA or an aggregate score from multiple may be restricted to certain cancers. shRNAs targeting the same gene (Materials and Methods), we The previously reported mutual exclusivity of BRCA1/2 muta- identified 835 essential genes in either CCNE1-amplified and/ tions and CCNE1 amplification (9, 10) has not been validated in or overexpressing cancer cell lines (Table S1). We then con- an independent dataset. We, therefore, stratified tumors from the sidered four additional factors as evidence of significant bi- Australian Ovarian Cancer Study (n = 194) by BRCA1/2 germ-line ological relevance to further filter candidate genes (Fig. 2A) mutation (13), somatic mutation, or methylation status (14) and and identified those genes that were (i) among the top-ranked accurately measured CCNE1 copy number by quantitative PCR shRNA hits, (ii) located in recurrent amplicons reported by (qPCR) (Fig. 1B). Assessment of copy number by qPCR provides TCGA (9) and therefore, likely to be tumor drivers, (iii) coexpressed with CCNE1 (Fig. S1 and Table S2), and (iv) located a more accurate measure of the extent of copy number change (2) fi than microarray-based estimates used in prior studies (9). Al- in pathways signi cantly enriched among hits (Gene Go analysis) (Fig. S2). A total of 115 genes met at least one of our though low-level CCNE1 gain and BRCA1/2 mutation were ob- additional selection criteria (Fig. S3). High confidence hits, served, complete mutual exclusivity was seen between high-level fi = fi > meeting three or more ltering criteria (n 25), are shown CCNE1 ampli cation (log2 ratio 2; approximately eight in Fig. 2B. copies per genome) and BRCA1/2 germ-line mutations (Fisher Importantly, CCNE1 and CDK2, as well as other associated < test P value 0.01). Although the study was not adequately cell cycle genes, were ranked highly in our analysis, validating the powered for comparison with somatically mutated or methyl- experimental approach and supporting CDK2 as a key thera- ated samples, the same pattern was observed. Our findings suggest peutic target in CCNE1-amplified tumors (Fig. 2B). Other top- that there is a functional difference between low- and high-level ranked hits included TPX2, a centromeric protein that maintains CCNE1 copy number states and that there is a threshold of mitotic spindle integrity and genome stability (17). We have CCNE1 amplification where BRCA1/2 inactivation is unlikely previously shown that the TPX2 locus, located at 20q11, is fre- to co-occur. quently coamplified with CCNE1 (5). Our findings support the

A Ovarian serous CCNE1 B * Bladder urothelial CCNE1, URI1 5 ** 0.5 Stomach adenocarcinoma C19orf12, CCNE1, URI1 0.5 - 2 Uterine corpus endometrioid CCNE1 4 2 Lung squamous cell CCNE1, URI1 All lung cancers CCNE1, URI1 3 All epithelial cancers CCNE1 2 Breast invasive 7 genes

Lung adenocarcinoma 6 genes number ratio copy 1 2 Colorectal 119 genes Colon adenocarcinoma 21 genes log 0 Glioblastoma multiforme CCNE1 -1 All glial 5 genes CCNE1 All cancers CCNE1 -2 0 5 10 15 20 25 BRCA2 Frequency of Amplification (%) BRCA1 BRCA1 BRCA2 BRCA1 BRCA1/2 GL GL SOM SOM WT METH

Fig. 1. (A) Pan-cancer copy number analysis of 6,547 tumor samples comprising 22 cancer types from TCGA. Frequency of high-level amplification of peak regions of copy number change incorporating CCNE1 is shown for cancer types with amplification. Data were obtained from the TCGA Copy Number Portal using all available data as of February of 2013. Name or total number of genes including CCNE1 within significant peak regions of amplification is indicated. (B) CCNE1 copy number assessed by qPCR in primary tumor samples from the Australian Ovarian Cancer Study (n = 193) stratified by WT, germ-line (GL), or somatic (SOM) BRCA1/2 mutation or methylation (METH) status. Bars indicate mean and SD. t test. *P value < 0.05; **P value < 0.01.

19490 | www.pnas.org/cgi/doi/10.1073/pnas.1314302110 Etemadmoghadam et al. Downloaded by guest on September 30, 2021 Candidate essential genes amplification) (12), were found in our analysis of the shRNA A Genes in n = 835 amplified region screen data. We also noted that genes involved in DNA damage response (DDR) were identified as essential genes in the screen, CCNE1 including BRCA1, XRCC2 (Fig. 2B), and ATR (Fig. S3). These CCNE1 high amplified findings suggest that chromosomal segregation and DDR mech- 125 expression n = 474 n = 486 anisms may be specific vulnerabilities of CCNE1-amplified or overexpressing cells. Finally, Ubiquitin-like modifier activating en- zyme 1 (UBA1) was among the top 25 hits from the screen (Fig. CCNE1 2B). We also observed that other members of the ubiquitin CDK2 Genes co-expressed with CCNE1 pathway, including Ubiquitin B (UBB), Ubiquitin C (UBC), and Ring-Box Protein 1 (RBX1), were among the top 115 ranked genes in the initial screen (Fig. S3), and they were, therefore, included in the validation studies. Genes in enriched pathways Top Ranked Hits Acute Effects of Gene Suppression. We sought to validate hits from the shRNA screen using an orthogonal siRNA platform. Com- pared with the shRNA screen, shorter-term (5 d) siRNA experiments provide additional information on the acute effects of gene suppression on cell viability. We screened two cells lines, SK-OV-3 (CCNE1-unamplified cells) and OVCAR-3 (CCNE1-amplified B cells), against a boutique siRNA library of 115 highly ranked hits from the shRNA screen (Fig. 2B and Fig. S3). Additionally, we included 27 candidate genes based on their biological relevance (Fig. S4). Selected candidates included cell cycle and DDR (TCGA) genes in addition to genes involved with processing and degradation of CCNE1 protein (15). Suppression of PLK1 was CCNE1 used as a positive (death) control (18). Cells were transfected High Expression Amplified with siRNA, and viability was measured 5 d after transfection. fi CCNE1 Volcano plots of P value signi canceagainsttheeffectoncell CCNE1 viability are shown for each cell line in Fig. S5, and all data are provided in Table S3. Located in an Amplicon (TCGA) Number Selection Criteria Met KS p-value KS p-value Essential Essential Co-expressed with In Significant GeneGo Pathway shRNA Hit Ranking Top Gene* Cell Cycle DNA Damage Response/Repair Gene* DNA Damage Response/Repair 2nd Best p-value 2nd Best p-value The cell viability ratio between OVCAR-3 and SK-OV-3 High Confidence Hits fi CDK2 D 0.0171 p<0.001 D p<0.001 p<0.001 YDD5 ll plotted against the P value signi cance for OVCAR-3 cells CCNE1 C 0.0088 0.0269 C 0.0192 0.1069 Y NA C 4 ll highlights significant hits with highest specificity to the CCNE1- ACAT2 CA 0.0039 p<0.001 CA 0.0138 p<0.001 CA CA 4 amplified cell line (Fig. 3A). Among these genes were CCNE1, CSE1L SE 0.1057 0.0153 SE 0.0133 p<0.001 SE SE 4 CDK2, and BRCA1 and other genes involved with cell division BRCA1 R 0.059 0.0096 0.1108 0.1836 RR 3 ll D CCNA2 0.0878 0.0645 C 0.0263 0.0112 CC 3 ll and DNA damage response and repair (ATM, CHEK1, and CDC42 D 0.0235 p<0.001 D 0.0099 0.007 D 3 SMC2). The greatest impact on cell viability in CCNE1-amplified CHD2 H p<0.001 0.0513 0.0533 0.7309 YH3 cells was associated with suppression of genes involved in the DDX17 D 0.0093 0.0028 D 0.0012 p<0.001 D 3 ubiquitin pathway, including UBA1, UBC, and RBX1, in addition DUSP16 U p<0.001 0.0087 U 0.0035 0.0334 U 3 ENPP2 NP p<0.001 0.0046 NP 0.0031 0.0011 NP 3 to CUL3 and FBXW7, which were included in the siRNA screen HNRNPA3 N 0.0833 0.017 N p<0.001 0.0029 Y 3 based on their roles in CCNE1 processing (15). IARS2 R p<0.001 0.0049 R p<0.001 0.0171 R 3 Although CCNE1 and CDK2 rank among the most significant MYC 0.5835 0.8201 YC 0.213 0.014 YYC3 hits in the OVCAR-3 cell line, the magnitude of the effect on cell PSMA5 M 0.0443 0.0204 M 0.0083 p<0.001 M 3 fi RRM1 R 0.012 p<0.001 R p<0.001 0.0159 R 3 viability was limited (Table S3). This nding is consistent with SLC35A3 C3 0.0293 0.0054 C3 p<0.001 0.0265 C3 3 our previous studies that show a greater effect of gene sup- SMC2 M 0.021 0.0072 M 0.0352 0.0051 M 3 ll pression using siRNA (5) or CDK-specific inhibitors (7) in clo- SPATA6 AT 0.0189 0.1308 AT p<0.001 0.0194 AT 3 nogenic survival assays compared with short-term viability assays. SRBD1 RB 0.0246 0.0496 RB 0.0188 0.1869 Y 3 TPX2 X2 0.0036 p<0.001 0.0766 0.0746 YX2 3 ll Similarly, for BRCA1, we showed a greater effect of inhibition TUBB B 0.0026 0.0018 B 0.0015 p<0.001 B 3 of BRCA1 in clonogenic survival assays (∼50% reduction) than GENETICS UBA1 BA 0.0325 0.0049 BA 0.5509 0.02 BA 3 observed in the siRNA boutique screen (Fig. 3B and Fig. S5). VCP CP p<0.001 0.0049 CP p<0.001 p<0.001 CP 3 D Knockdown of each gene transcript was validated by real-time XRCC2 0.0366 0.0385 R 0.0211 0.0313 YR 3 D qPCR (Fig. S6). Fig. 2. (A) Venn diagram of candidate genes essential for the survival of CCNE1-amplified (n = 474) and overexpressing (n = 486) cell lines identified Targeting Homologous Recombination and the Proteasome. De- in the shRNA synthetic lethal screen. Candidates were further filtered to pendence on BRCA1 suggests that intact HR function is required include genes that were present in commonly amplified regions in ovarian for the survival of CCNE1-amplified tumor cells. Fanconi Ane- tumors, coexpressed with CCNE1, present in significantly enriched gene mia (FA) pathway members coordinate multiple DNA repair pathways, or among top-ranking shRNA hits. (B) Top 25 ranking genes an- mechanisms, including HR. Recently, a cell-based screen of over fi notated by inclusion criteria. Statistical signi cance of ranking by second- 16,000 compounds identified the proteasome inhibitor bortezo- best scoring shRNA or a composite score of all shRNAs (KS statistic) given (Materials and Methods). *Go term processes: cell cycle, DNA repair, or re- mib as a potent inhibitor of the FA pathway and double-strand sponse to DNA damage. break repair by HR (19). It is thought that disruption of protein degradation by bortezomib either interferes with or is a requirement of FA pathway activity. The mutual exclusivity ob- view that cooperative amplification of 19q12 and 20q11 is im- served between HR pathway dysfunction and CCNE1 amplification portant to the genesis and/or maintenance of CCNE1-amplified and the dependence on genes involved with protein degradation tumors. No other genes within the 19q12 amplicon, including suggested that CCNE1-amplified tumors may show selective sen- URI1 (which was previously suggested to be a target of gene sitivity to bortezomib. We assessed ovarian cell line sensitivity to

Etemadmoghadam et al. PNAS | November 26, 2013 | vol. 110 | no. 48 | 19491 Downloaded by guest on September 30, 2021 A -2.5 B CHAF1A SNRPF 0.25 CUL3 -2.0 UBA1 CKAP5 *** SMC2 UBC RBX1 * -1.5 PLK1 120 UBB CCNK CDC5L *** SK-OV-3 LSM5 TPX2 LSM4 100 OVCAR-3 -1.0 0.50 RPA3 RRM1 TUBB SRSF2 80 PSMA5 SIN3A CCNE1 NRBP2 CHEK1 -0.5 CAPN2RB1 FBXW7 60 DDX17 BPTFHNRNPA3 ZNF383 ANKRD17YWHAHCCND1CORO1CCDC25APAX8BRCA2 ATR COL9A2CHEK2BRCA1 CDK2 TMEM48TOP2A CDK5 40 HERC5RAD50 ATM COL14A1C9TNFRSF11ARALGDS 0.0 NUP153CDC42AKT2 CDK4 VCPOR2AK2 ABCG8 RBBP8 LGALS2 20 PREPL MRPL12CUL1CAPZB MYC PCSK6 SLC38A2 CDC23 no siRNA to colonies % EIF6 DDB1 0 CPAMD8 CDK1 viability (OVCAR-3 / SK-OV-3) 0.5 RNF8 2 NS NS CDK9 log CDK2 1.0 CDK2 BRCA1 BRCA2 BRCA1 BRCA2 CSE1L p < 0.05 1.5 0 -8-6-4-2 -20-18-16-14-12-10

log2 p-value OVCAR-3

Fig. 3. (A) Cells were transfected with a boutique siRNA library against 142 candidate genes, and the effect on cell viability was measured 5 d after transfection. Significance (t test P value) of hits in the OVCAR-3 (CCNE1-amplified) cell line plotted against the viability ratio of OVCAR-3 to SK-OV-3 (unamplified) highlights significant hits specific to OVCAR-3. Average data from duplicate wells across three independent experiments are shown (n = 3). The vertical dotted line is at P value = 0.05. (B) Clonogenic survival after siRNA transfection in SK-OV-3 (unamplified) and OVCAR-3 (CCNE1-amplified) ovarian cell lines. Average percentage of discrete colonies formed after 7 d relative to no siRNA controls is shown (n = 3 independent experiments performed in triplicate). Statistical significance (t test) was calculated by comparison with nonsilencing (NS) siRNA in the same cell line. *P value < 0.05; ***P value < 0.001. Error bars indicate SEM.

bortezomib in a panel of 10 cell lines (Fig. 4). FUOV-1 and with low-level CCNE1 gain, did not show heightened sensitivity OVCAR-3, which have high-level CCNE1 amplification and to bortezomib. Interestingly, Kuramochi and IGROV-1, with expression, showed the greatest sensitivity to bortezomib. A2780, reduced sensitivity to bortezomib, harbor mutations in BRCA2 which lacks CCNE1 amplification but strongly expresses CCNE1, and BRCA1, respectively (20). We also examined two cell lines was also among the most sensitive lines. By contrast, Kuramochi, derived from the OVCAR-3 parental line that are resistant to two CDK2-specific inhibitors PHA-533533 and dinaciclib (OVCAR-3- R1 and -RD1, respectively) (7). We found that both lines maintained sensitivity to bortezomib, suggesting a specificmech- 60 anism of resistance to CDK inhibitors in these cell lines. 50 Discussion 40 BRCA1/2 mutation is typically associated with platinum sensi- 30 tivity and favorable clinical outcome (13). The absence of BRCA pathway disruption from CCNE1-amplified cancers may partly 20 explain the relatively poor outcome observed in CCNE1- fi 10 ampli ed tumors (9). Mutual exclusivity of oncogenic muta- Bortezomib IC50 (nM) IC50 Bortezomib tions in genes in the rat sarcoma viral oncogene homolog 0 (RAS) signaling pathway, seen in low-grade serous ovarian Expression cancer (21) and other solid cancers (22), seems to occur, be- Copy Number cause there is no selective advantage of compound mutations. By contrast, we found that BRCA1 suppression is not tolerated in cell lines that harbor CCNE1 amplifications. The specificre-

A2780 quirement of BRCA1 compared with BRCA2 may relate to its FUOV-1 OAW-42 SK-OV-3 IGROV-1 wider roles in DNA repair as well as cell cycle regulation and OVCAR-5 OVCAR-8 OVCAR-4 OVCAR-3 Kuramochi checkpoint activation (11). OVCAR-3-R1 CCNE1 overexpression promotes unscheduled S-phase entry, OVCAR-3-RD1 CCNE1 Copy Number CCNE1 Expression disrupted DNA replication, and genomic instability (15), po- tentially rendering cells dependent on intact HR repair path- Unamplifed Low ways. We also observed dependencies on genes involved in Gained High processing of CCNE1 and other components of protein degra- Amplified dation pathways. Synthetic lethality is seen as an important ap- Fig. 4. Ovarian tumor cell line sensitivity to bortezomib ranked by average proach to the development of new cancer therapeutics, because

72-h cytotoxicity assay IC50 value (n = 3 independent experiments performed it suggests treatments that are likely to offer a wide therapeutic in triplicate). Error bars indicate SEM. CCNE1 copy number status was de- index (23). Indeed, our findings suggest that the proteasome termined by qPCR, where copy number gain and amplification are defined inhibitor bortezomib, either through attenuation of HR or other > > CCNE1 as a log2 ratio to normal 0.5 and 2.0, respectively. gene expression essential proteasome functions in CCNE1-amplified cells, offers of each cell line above (high) or below (low) the median value of 10 parental lines is indicated. The OVCAR-3-R1 and -RD1 sublines were derived from a unique therapeutic approach in HGSCs and possibly other OVCAR-3 and are resistant to CDK2 inhibitors PHA-533533 and dinaciclib, solid cancers. Additionally, the lack of cross-resistance to bor- respectively (7). tezomib in cells previously rendered partially resistant to CDK2

19492 | www.pnas.org/cgi/doi/10.1073/pnas.1314302110 Etemadmoghadam et al. Downloaded by guest on September 30, 2021 inhibitors (OVCAR3-R1/RD1) suggests that sequential treat- shRNA Screen Data. Cell line copy number data were obtained from the ment may be effective. Cancer Cell Line Encyclopedia (20). CCNE1 copy number and gene expression HGSC patients with high-level CCNE1 amplification represent status was assigned to each cell line with midrange samples removed from > CCNE1 an urgent unmet need given their high risk of treatment failure and the analysis. Cell lines with a log2 copy number ratio 0.3 over the locus were defined as amplified (n = 23), and cell lines with a log2 copy low probability of response to PARP inhibitors because of absence number ratio < 0overtheCCNE1 locus were defined as unamplified (n = 43). of BRCA1/2 pathway dysfunction. Bortezomib is currently used in Cell lines with CCNE1 gene expression greater than median + 1SD(n = 15) multiple myelomas and mantle cell lymphoma, but it has not shown were classified as CCNE1 high expression, whereas cell lines with CCNE1 gene significant activity in other solid cancers, including ovarian cancer expression less than median (n = 41) were classified as CCNE1 low expression. (24). The low frequency of high-level amplification of CCNE1 in Microarray data from shRNA experiments was obtained from the In- HGSCs may require specific patient selection to observe a thera- tegrative Genomics Portal (http://www.broadinstitute.org/igp). peutic benefit. Here, a mutational interaction observed in patient Data were analyzed using the GenePattern module ScorebyClassComp samples is explained molecularly, and a unique treatment ap- and GENE-E software (28). The weight of evidence statistic was used for class CCNE1 fi fi CCNE1 proach is defined. discrimination between -ampli ed and unampli ed cells and high- and low-expressing cell lines (16). Gene lists were created by collapsing Materials and Methods shRNA ranks from each comparison, and then, they were ranked by significance to identify top hits as described previously (16). First, we selected the Ethics Statement. The Australian Ovarian Cancer Study was approved by the top 300 genes based on the second-best ranking shRNAs, and second, we Human Research Ethics Committees at the Peter MacCallum Cancer Centre, selected the top 300 genes assessed using the Kolmogorov–Smirnov (KS) Queensland Institute of Medical Research (QIMR), University of Melbourne statistic, which uses a composite score for all shRNAs against each gene (28). and all participating hospitals. Written informed consent was obtained from The union of both analyses identified 474 essential genes in CCNE1-ampli- all participants in this study. fied cell lines and 486 essential genes in CCNE1 high-expressing cell lines (Fig. 2A and Table S1). Because each gene was targeted by multiple independent CCNE1 Pan-Cancer Analysis of Copy Number. Peak regions of copy number shRNAs (median n = 5 per gene), statistical assessment of essentiality may be CCNE1 fi change at the locus identi ed by GISTIC (25) were obtained from the affected by the number of shRNAs used per gene. For example, a higher TCGA Copy Number Portal (http://www.broadinstitute.org/tcga) using all avail- number of shRNAs against CDK2 compared with CCNE1 and BRCA1 (n = 12, able data as of February of 2013 (dataset: 2013-02-21 stddata__2013_02_03). n = 4, and n = 7, respectively) may, in part, explain the higher ranked sig- Analysis included a total of 6,547 tumor samples comprising 22 cancer types: nificance of CDK2 in our analysis (Table S1). n = n = breast invasive adenocarcinoma ( 891), glioblastoma multiforme ( The candidate gene list was further refined using additional criteria as n = 563), ovarian serous cystadenocarcinoma ( 559), kidney renal clear cell evidence of significant biological relevance, including if they were (i) among n = n = carcinoma ( 493), uterine corpus endometrioid carcinoma ( 492), the top 10 shRNA hits ranked by either the second-best or KS method, (ii) n = n = thyroid carcinoma ( 430), colon adenocarcinoma ( 413), lung adeno- located in recurrent minimal regions of amplification reported by TCGA (9), n = n = carcinoma ( 403), lung squamous cell carcinoma ( 358), head and neck (iii) coexpressed with CCNE1 (see above), or (iv) located in pathways signif- n = n = squamous cell carcinoma ( 322), stomach adenocarcinoma ( 237), icantly represented among all hits (Gene Go analysis). In total, 115 genes met n = n = cutaneous melanoma ( 236), brain lower-grade glioma ( 220), prostate at least one of our additional selection criteria (Fig. 2B and Fig. S3) and were n = n = adenocarcinoma ( 177), rectum adenocarcinoma ( 162), bladder uro- selected for validation studies. thelial carcinoma (n = 150), kidney renal papillary cell carcinoma (n = 117), n = cervical squamous cell carcinoma ( 114), liver hepatocellular carcinoma Cell Lines. Ovarian cell lines were obtained from the National Cancer Institute n = n = n = ( 97), kidney chromophobe ( 66), sarcoma ( 29), and diffuse large Repository and fingerprinted using short tandem repeat markers (29) to n = B-cell lymphoma ( 18). confirm identity against the Cancer Genome Project database (Wellcome

Trust Sanger Institute). Cells were maintained at 37 °C and 5% (vol/vol) CO2 BRCA1/2 and CCNE1 Status in Primary Ovarian Tumor Samples and Cell Lines. in RPMI 1640 containing 10% (vol/vol) FCS, with transfection and drug We have previously published our analysis of germ-line BRCA1/2 status (13) and sensitivity assays performed in the absence of antibiotics. Cell lines were somatic analysis (14) of tumor samples from women enrolled in the Australian confirmed to be mycoplasma-negative before siRNA studies. Ovarian Cancer Study. TCGA estimated the frequency of CCNE1 gain to be ∼ BRCA1/2 8% and 26% in -altered and WT cases, respectively (9). We calcu- siRNA Studies. A boutique On-Target Plus siRNA library was obtained from lated that analyses of ∼80 BRCA1/2-altered and 80 WT cases would give 80% Dharmacon (Thermo Fisher Scientific) in 384-well plates containing 142 power (sensitivity) to detect a difference between the two groups, where α = candidate genes, a nonsilencing control, and a positive (death) control (PLK1). 0.05 (probability of a false-positive result). Our final cohort (n = 193) included The library was hydrated and diluted to 1 μM in siRNA buffer (Dharmacon; 81 BRCA1/2 WT and 112 BRCA1/2-altered cases (Fig. 1B). The BRCA1/2-altered Thermo Fisher Scientific). Cells were reverse transfected with DharmaFECT group included samples with germ-line BRCA1 mutations (n = 52), germ-line lipid reagents (Thermo Fisher Scientific) to a final concentration of 40 nM BRCA2 mutations (n = 29), somatic BRCA1 mutations (n = 5), somatic BRCA2 siRNA using SciClone ALH 3000 (Caliper Life Sciences) and BioTek 406 mutations (n = 4), and BRCA1 methylation (n = 22). CCNE1 copy number rel- (BioTek) liquid handling robotics. Cell transfection densities were selected to ative to normal female reference DNA (Novagen) and gene expression in cell achieve confluence 5 d after transfection (120 h; 800 cells per well for

lines was determined using qPCR and previously described methods (5). OVCAR-3; 500 cells per well for SK-OV-3). Selected transfection conditions GENETICS allowed for efficient siRNA transfection with no impact on cell viability TCGA SNP and Gene Expression Data. Affymetrix SNP 6.0 and hthgu133a gene (OVCAR-3, 0.04 μL DharmaFECT1 per well; SK-OV-3, 0.06 μL DharmaFECT2 expression data were obtained for 157 serous tumors from TCGA (www. per well). During optimization experiments, nuclear localization of siGLO cancergenome.nih.gov). All SNP CEL files were normalized in a single batch red RNA duplex (Dharmacon) was used to monitor transfection efficiency by using the R package aroma.affymetrix and then segmented using the cir- fluorescence microscopy 24–48 h after transfection. cular binary segmentation algorithm to improve the signal-to-noise ratio Cells were transfected in duplicate wells in three independent experi- (26). CCNE1 copy number was estimated using the mean segment value, and ments. Cell viability was then assessed using the Cell Titer Glo luminescent amplification was called for samples where the mean segment log2 copy assay (Promega) on the Synergy H4 high-throughput multimode microplate number ratio value was greater than 0.3. Recurrently amplified genes reader (BioTek). Average cell viability data for each gene were normalized to identified in the TCGA dataset have been previously published (9). Gene the average signal from control wells containing lipid only (n = 12 per plate).

expression CEL files were normalized using the GCRMA package in R (27). Significance of change in cell viability (log2-transformed signal) compared Pearson correlation coefficient was computed between CCNE1 and all other with control wells was calculated using a t test (n = 3). Clonogenic survival probes in the genome. Genes that had an false discovery rate (FDR)-cor- assays and real-time qPCR were performed as described previously (5). rected P value <0.05 and a correlation coefficient >0.25 or <−0.25 were consider to be coexpressed or anticorrelated with CCNE1, respectively. We Sensitivity to Bortezomib. Bortezomib was obtained from Millennium Phar- identified 501 genes that were coexpressed with CCNE1 (Table S2). Pathway maceuticals, and drug sensitivity was determined using the CellTiter 96 analysis of genes coexpressed with CCNE1 using GeneGo (Thomson Reuters) Aqueous Non-Radioactive Cell Proliferation Assay (Promega) as described

revealed an enrichment of gene lists involved with cycle and DNA damage previously (5). IC50 dose was approximated by ﬁtting a four-parameter dose– response pathways (Fig. S1). response curve (Hill equation) using Prism 6 (GraphPad Software).

Etemadmoghadam et al. PNAS | November 26, 2013 | vol. 110 | no. 48 | 19493 Downloaded by guest on September 30, 2021 ACKNOWLEDGMENTS. The authors acknowledge assistance from Daniel Genotyping of AOCS patient samples was supported by Ovarian Cancer Thomas and Yanny Handoko in conducting siRNA experiments in the Victorian Research Program of the US Department of Defense Grants W81XWH-08-1- Centre for Functional Genomics, Peter MacCallum Cancer Centre. The Australian 0684 and W81XWH-08-1-0685; Cancer Australia and National Breast Cancer Ovarian Cancer Study (AOCS) acknowledges the cooperation of the participat- Foundation Grants ID509303, CG-08-07, and ID509366; the Peter MacCal- ing institutions in Australia and the contribution of the study nurses, research lum Cancer Centre Foundation; and the Cancer Council Victoria. The assistants, and all clinical and scientiﬁc collaborators. We thank all of the women Victorian Centre for Functional Genomics is funded by the Australian who participated in the study. This study was funded by National Health and Cancer Research Foundation and the Victorian Department of Industry, Medical Research Council (NHMRC) Project Grant APP 1042358, Cancer Innovation and Regional Development. The Australian Phenomics Network is Australia Grant APP 1004673, and US National Institutes of Health Grant supported by funding from the Australian Government’s Education Investment U01 CA176058. The AOCS was supported by US Army Medical Research Fund through the Super Science Initiative, the Australasian Genomics Technol- and Materiel Command Grant DAMD17-01-1-0729, the Cancer Council Tasmania, ogies Association, the Brockhoff Foundation, and the Peter MacCallum the Cancer Foundation of Western Australia, and NHMRC Grant ID400413. Cancer Centre Foundation.

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