King’s Research Portal DOI: 10.1038/s41467-018-03283-z Document Version Publisher's PDF, also known as Version of record Link to publication record in King's Research Portal Citation for published version (APA): Patel, N., Weekes, D., Drosopoulos, K., Gazinska, P., Noel, E., Rashid, M., Mirza, H., Quist, J., Brasó-Maristany, F., Mathew, S., Ferro, R., Pereira, A. M., Prince, C., Noor, F., Francesch-Domenech, E., Marlow, R., de Rinaldis, E., Grigoriadis, A., Linardopoulos, S., ... Tutt, A. N. J. (2018). Integrated genomics and functional validation identifies malignant cell specific dependencies in triple negative breast cancer. Nature Communications, 9(1), [1044]. https://doi.org/10.1038/s41467-018-03283-z Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. General rights Copyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights. •Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research. •You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 02. Oct. 2021 ARTICLE DOI: 10.1038/s41467-018-03283-z OPEN Integrated genomics and functional validation identifies malignant cell specific dependencies in triple negative breast cancer Nirmesh Patel1,2, Daniel Weekes1,2, Konstantinos Drosopoulos3, Patrycja Gazinska1,2, Elodie Noel1,2, Mamun Rashid1,2, Hasan Mirza1,2,4, Jelmar Quist1,2,4, Fara Brasó-Maristany1,2, Sumi Mathew1,2, Riccardo Ferro1,2, Ana Mendes Pereira1,2, Cynthia Prince1,2, Farzana Noor1,2, Erika Francesch-Domenech1,2, Rebecca Marlow1,2, Emanuele de Rinaldis1,2,5, Anita Grigoriadis 1,2,4, Spiros Linardopoulos3,6, Pierfrancesco Marra1,2 & Andrew N.J. Tutt 1,2,3 1234567890():,; Triple negative breast cancers (TNBCs) lack recurrent targetable driver mutations but demonstrate frequent copy number aberrations (CNAs). Here, we describe an integrative genomic and RNAi-based approach that identifies and validates gene addictions in TNBCs. CNAs and gene expression alterations are integrated and genes scored for pre-specified target features revealing 130 candidate genes. We test functional dependence on each of these genes using RNAi in breast cancer and non-malignant cells, validating malignant cell selective dependence upon 37 of 130 genes. Further analysis reveals a cluster of 13 TNBC addiction genes frequently co-upregulated that includes genes regulating cell cycle check- points, DNA damage response, and malignant cell selective mitotic genes. We validate the mechanism of addiction to a potential drug target: the mitotic kinesin family member C1 (KIFC1/HSET), essential for successful bipolar division of centrosome-amplified malignant cells and develop a potential selection biomarker to identify patients with tumors exhibiting centrosome amplification. 1 Breast Cancer Now Research Unit, King’s College London, London SE1 9RT, UK. 2 School of Cancer and Pharmaceutical Sciences, King’s Health Partners AHSC, Faculty of Life Sciences and Medicine, King’s College London, London WC2R 2LS, UK. 3 The Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London SW7 3RP, UK. 4 Cancer Bioinformatics, King’s College London, London SE1 9RT, UK. 5 Precision Immunology Cluster, Sanofi, 640 Memorial Drive, Cambridge, MA 02149, USA. 6 Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UK. These contributed equally: Pierfrancesco Marra, Andrew N. J. Tutt. Correspondence and requests for materials should be addressed to A.N.J.T. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:1044 | DOI: 10.1038/s41467-018-03283-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03283-z – riple negative breast cancers (TNBCs) are difficult to treat normal breast epithelium samples14 16. All clinico-pathological Tand lack expression of the validated breast cancer ther- features of the cohort are provided in Supplementary Data 1.We apeutic targets: estrogen (ER), progesterone (PR), and obtained gene-centric copy number levels, frequency and focality human epidermal growth factor 2 (HER2) receptors1. TNBCs are of gene copy number changes as well as the gene expression from heterogeneous2 with substantial numbers of patients in subgroups this cohort (Fig. 1a). The data were integrated with analyses of that have high risk of early metastatic relapse commonly resistant publicly available databases such as COSMIC17, the membra- to systemic therapy. Despite frequent resistance, chemotherapy is nome18, the druggable genome19, secretome20, CAN genes21, and the only widely accepted systemic therapy option for these kinome22 (Supplementary Data 2). All the above data were col- patients, highlighting the need to better understand the under- lated in the Target ID data platform that was used as a foundation lying biology and identify tumor cell-specific therapy targets for for the application of a pre-specified selection algorithm for drug discovery or “repositioning” of known therapies. putative addiction genes in TNBC. Identification of tumor addictions (dependence on a gene for The Target ID data platform informed two complementary proliferation and survival) has in the past led to the development approaches to gene selection for functional validation (mRNA of novel therapies, notably the discovery of ERBB2 amplification overexpression and gene amplification/ mRNA expression and overexpression, now targeted by a number of therapies in correlation) with the aim being to minimize bias and limitations breast cancer3. Despite progress in characterizing the genomic inherent to any single analytical procedure. First, a copy number- – landscape of breast cancer4,5 and TNBC specifically2,6 8, targe- dependent gene expression analysis selected 1978 candidate genes table biological dependencies remain elusive and poorly char- amplified in >10% of TNBCs with a gene copy number/gene acterized. With the exception of clonally dominant mutations in expression correlation (r2 > 0.3, p < 0.01, Spearman’s rank corre- TP53, TNBCs demonstrate a high degree of inter-tumor and lation). We then linked the candidate genes to features, included intra-tumor heterogeneity at the mutational level with each driver in the Target ID data platform, which are known to be relevant to mutation only present in a subset of tumors and clones within hallmarks of malignancy in a weighted scoring system (Fig. 1a any individual tumor9. and Supplementary Data 3). The top 85 genes were taken forward TNBCs have a high frequency of chromosomal instability for functional validation. resulting in variable copy number state and levels of gene Second, a complementary gene expression-centered analysis expression7,10. Genes that are found in amplified regions and are was used as there is evidence that variations in the expression of highly expressed, may be drivers of important “hallmarks” of tumor addiction genes may also occur in the absence of in cis malignancy11 and potentially represent essential tumor addic- CNAs through multiple mechanisms, for example through tions. A number of high-throughput loss of function screening epigenetic regulation23. For the gene expression-centered analysis, studies have identified gene addictions in cellular models of we identified 1001 genes whose average expression in our TNBC cancer including breast cancer models4,5,12 but functional vali- cohort was >2-fold higher than normal breast epithelium dation has been limited and studies have rarely been informed by controls. As anticipated by the inclusion of CNA correlated evidence of upregulation of gene copy number or mRNA in large elevated gene expression in the first approach there was a numbers of patient tumors. Therefore, the main aim of this study substantial overlap with 45 of the genes from the second is to identify and validate recurrently amplified genes as being approach which were taken forward for functional validation. important for malignant phenotypes in TNBC. We perform a An additional 45 genes identified by this second approach were pre-specified integrative computational “driver” identification selected based exclusively on gene annotation and literature and RNAi-based functional validation approach, taking into review. This manually curated filtering sought to identify genes account both the copy number landscape and whole genome already shown to drive a tumor phenotype in other cancer models expression state in individual tumors, using a large discovery or being involved in biological pathways known to be key in cohort