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Breast cancer metastases is one of the leading causes of cancer related death in women worldwide. Our fundamental understanding of treatment resistance, disease progression and metastasis in cancer has greatly improved with the advent of -omic technologies. However, a deeper understanding of the molecular complexity, in particular, in the context of brain metastasis is still lacking. In this thesis, through interrogation of high through-put transcriptomic, genomic and other data, clinically relevant molecular mediators of endocrine resistant and brain metastatic breast cancer are identified.Estrogen receptor positive (ER+) cancer accounts for ≈70% of all breast cancer cases, with endocrine resistance a major clinical problem. In results Chapter 2, elucidation of the role p160 proteins, SRC-1 and AIB1, play in endocrine resistance is given. Specifically, a previously under appreciated novel role for Nucleosome Remodeling and Deacetylase (NuRD) complex mediated gene repression is demonstrated, along with identification of PARP1 as common to p160 interactomes in endocrine resistant cells. Moreover, data here, underscores the necessity to better understand the role of epigenetic aberrations in breast cancer metastases, in particular in the context of treatment resistance and clinically relevant receptor discordance.In Chapter 3 and 4, along with intrinsic molecular subtype switching observed in brain metastases, most prominent in ER+ patients; an unprecedented subtype specific analysis of molecular alterations in brain metastatic breast cancer is provided, where collectively, this study will serve as a critical reference for future transcriptomic studies of brain metastases. Lastly, in Chapter 4, DNA repair deficiency is found to be enriched in brain metastatic disease. Compelling pre-clinical data indicates homologous recombination deficiency (HRD) specifically may be a key therapeutic vulnerability to exploit for treatment of breast cancer brain metastases, and should inform future clinical studies.In conclusion, work presented here comprehensively characterises novel molecular mediators of endocrine resistant and brain metastatic breast cancer with potential therapeutic implications for treatment.

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere. We want to thank all members from the Brain Metastasis Group, F.X. Real, M. Malumbres, and M. Barbacid for critical discussion of the manuscript, the CNIO Core Facilities for their excellent assistance, Antonio Cebri a and Javier Klett for their excellent assistance in drug-screening, Sonsoles RodrıguezArıstegui for her excellent work with preparation of different compounds and Alexandra de Francisco, Yolanda Sierra, and Marıa de la Jara Felipe for their excellent work with animal preparation and imaging protocols. We also thank J. Massague (MSKCC) for some of the BrM cell lines. This work was supported by MINECO (SAF2017-89643-R, SAF2014-57243-R, SAF2015-62547-ERC) (M.V.), Fundacion FERO (IX FERO Grant for Research in Oncology) (M.V.), Fundacio La Marato de TV 3 (141) (M.V.), Melanoma Research Alliance (Bristol-Myers Squibb-Melanoma Research Alliance Young Investigator Award 2017 (https:// doi.org/10.48050/pc.gr.75716)) (M.V.), Beug Foundation (Prize for Metastasis Research 2017) (M.V.), Fundacion Ramon Areces (CIVP19S8163) (M.V.) and CIVP20S10662 (E.O.P.), Worldwide Cancer Research (19-0177) (M.V.), H2020- FETOPEN (828972) (M.V.), Cancer Research Institute (Clinic and Laboratory Integration Program CRI Award 2018 (54545)) (M.V.), AECC (Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.), LAB AECC 2019 (LABAE19002- VALI) (M.V.), ERC CoG (864759) (M.V.), Sophien-Stiftung zur Förderung der klinischen Krebsforschung (T.W.), Promedica Stiftung (T.W.), Stiftung fur ange- € wandte Krebsforschung (T.W.), Forschungskredit of the University of Zurich (FK-18-054) (T.W.), Betty and David Koetser Foundation for Brain Research (T.W.), Foundation for Applied Cancer Research in Zurich (T.W., M.W.), Comunidad de Madrid (S2017/BMD-3867 RENIM-CM and Y2018/NMT-4949 NanoLiver-CM) and European structural and investment funds (M.D.), ISCIII (PT20/00044) co-funded by FEDER “A way of making Europe” (M.D.), Ministero dell’Istruzione, dell’Universit a e della Ricerca-MIUR, “Dipartimenti di Eccellenza 2018-2022”, (D15D18000410001) (L.B. and P.C.), Science Foundation Ireland Frontiers for the Future Award (19/FFP/6443) (L.Y.), Science Foundation Ireland Strategic Partnership Programme, Precision Oncology Ireland (18/SPP/3522) (L.Y.), Breast Cancer Now Fellowship Award/ with the generous support of Walk the Walk (2019AugSF1310) (D.V.), La Caixa-Severo Ochoa International PhD Program Fellowship (LCF/BQ/SO16/52270014) (L.Z.), La Caixa International PhD Program Fellowship-Marie Sklodowska-Curie (LCF/BQ/DI17/11620028) (P.G-G), MINECO-Severo Ochoa PhD Fellowship (BES-2017-081995) (L.A-E.), AECC Postdoctoral Fellowship (POSTD19016PRIE) (N.P.), Boehringer Ingelheim Fonds MD fellowship (L.M.). The contribution of the Experimental Therapeutics Programme was supported by core funding from the Spanish National Cancer Research Center (CNIO). CNIO is supported by the ISCIII, the Ministerio de Ciencia e Innovacion, and is a Severo Ochoa Center of Excellence (SEV- 2015-0510). The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovacion and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.V. was named Ramon y Cajal Investigator (RYC2013-13365) and is member of EMBO YIP (4053). Sí

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.

Additional file 1: Supplemental Methods.

Additional file 2: Figure S1. Confirmation of LY2 ADAM22 CRISPR knockout cell line. (a) RFP expression confirming CRISPR /Cas9 mediated double stranded break and insertion of an RFP tagged HDR plasmid (b) FACS gating strategy based on high RFP expression to single cell sort CRISPR/Cas9 ADAM22 KO cells (right) from LY2 WT cells (left) (c) Confirmation of ADAM22 gene silencing in an LY2 ADAM22 KO clone (Clone H (C-H)) by PCR and Western blot analysis (n = 4). *** p

Additional file 2: Figure S1. Confirmation of LY2 ADAM22 CRISPR knockout cell line. (a) RFP expression confirming CRISPR /Cas9 mediated double stranded break and insertion of an RFP tagged HDR plasmid (b) FACS gating strategy based on high RFP expression to single cell sort CRISPR/Cas9 ADAM22 KO cells (right) from LY2 WT cells (left) (c) Confirmation of ADAM22 gene silencing in an LY2 ADAM22 KO clone (Clone H (C-H)) by PCR and Western blot analysis (n = 4). *** p

Additional file 1: Supplemental Methods.