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