Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2019 Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains Lambert, Jean-Philippe ; Picaud, Sarah ; Fujisawa, Takao ; Hou, Huayun ; Savitsky, Pavel ; Uusküla-Reimand, Liis ; Gupta, Gagan D ; Abdouni, Hala ; Lin, Zhen-Yuan ; Tucholska, Monika ; Knight, James D R ; Gonzalez-Badillo, Beatriz ; St-Denis, Nicole ; Newman, Joseph A ; Stucki, Manuel ; Pelletier, Laurence ; Bandeira, Nuno ; Wilson, Michael D ; Filippakopoulos, Panagis ; Gingras, Anne-Claude Abstract: Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers oppor- tunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs medi- ate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1. DOI: https://doi.org/10.1016/j.molcel.2018.11.006 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-160765 Journal Article Accepted Version Originally published at: Lambert, Jean-Philippe; Picaud, Sarah; Fujisawa, Takao; Hou, Huayun; Savitsky, Pavel; Uusküla- Reimand, Liis; Gupta, Gagan D; Abdouni, Hala; Lin, Zhen-Yuan; Tucholska, Monika; Knight, James D R; Gonzalez-Badillo, Beatriz; St-Denis, Nicole; Newman, Joseph A; Stucki, Manuel; Pelletier, Lau- rence; Bandeira, Nuno; Wilson, Michael D; Filippakopoulos, Panagis; Gingras, Anne-Claude (2019). Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains. Molecular Cell, 73(3):621-638.e17. DOI: https://doi.org/10.1016/j.molcel.2018.11.006 Interactome rewiring following pharmacological targeting of BET bromodomains Jean-Philippe Lambert1,12,‡, Sarah Picaud2,12, Takao Fujisawa3, Huayun Hou4,5, Pavel Savitsky2, Liis Uusküla-Reimand5,6, Gagan D. Gupta1¨, Hala Abdouni1, Zhen-Yuan Lin1, Monika Tucholska1, James D.R. Knight1, Beatriz Gonzalez-Badillo1, Nicole St-Denis1, Joseph A. Newman2, Manuel Stucki7, Laurence Pelletier1,4, Nuno Bandeira8,9,10, Michael D. Wilson1,4,11, Panagis Filippakopoulos2,3,*, Anne-Claude Gingras1,4,* 1 Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada. 2 Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, OX3 7DQ, UK. 3 Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, OX3 7DQ, UK. 4 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 5 Genetics and Genome Biology Program, SickKids Research Institute, Toronto, ON, Canada 6 Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia. 7 Department of Gynecology, University of Zurich, Wagistrasse 14, CH-8952 Schlieren, Switzerland 8 Center for Computational Mass Spectrometry, University of California, San Diego, La Jolla, California, 92093. 9 Dept. of Computer Science and Engineering, University of California, San Diego, La Jolla, California, 92093 10 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, 92093 11 Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Toronto, Canada 12 These authors have contributed equally ‡ Present address: Department of Molecular Medicine and Cancer Research Centre, Université Laval, Quebec, Canada; CHU de Québec Research Center, Quebec, G1V 4G2, Canada. ¨ Present address: Department of Chemistry and Biology, Ryerson University, Toronto, ON, M5B 2K3, Canada. Data deposition: The crystal structures reported in this paper have been deposited in the Protein Data Bank, http://www.pdb.org. Small-angle scattering data and models have been deposited to the SASBDB (http://www.sasbdb.org). Mass spectrometry data have been deposited in the ProteomeXchange partner Mass spectrometry Interactive Virtual Environment (MassIVE, http://massive.ucsd.edu). ChIP-seq data generated in this paper can be found online at ArrayExpress (http://www.ebi.ac.uk/arrayexpress; accession E-MTAB-5670). * Correspondance: E-mail : [email protected], Phone: (416) 586-5027, Fax: (416) 586-8869 E-mail : [email protected], Phone : +44 1865 617759, Fax : +44 1865 617575 Lead contact: Anne-Claude Gingras 1 Abstract Targeting bromodomains (BRDs) of the bromo-and extra-terminal (BET) family has offered opportunities for the therapeutic intervention of cancer and other diseases. However, there has not been a systematic study of the consequences of BET BRD inhibition on the interactions established by members of this important protein scaffolding family. Here, we profile the interactomes of BRD2, BRD3, BRD4 and BRDT during a time course of treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behaviour for the 603 unique interactors identified. We report a group of proteins that associate with BET BRDs through canonical and new binding modes that dissociate upon inhibitor treatment. We further establish two classes of extra-terminal (ET)-domain binding motifs that mediate acetylation- independent interactions, offering attractive possibilities for specific therapeutic modulation. Lastly, we identify an unexpected increase in several interactions following JQ1 treatment. These increased interactions allow us to define new negative functions for BRD3 in the regulation of rRNA synthesis and potentially Pol II-dependent gene expression that result in decreased cell proliferation. Together, our data outline the contributions of BET protein modules to their interactomes and allow for a better understanding of pharmacological rewiring in response to JQ1. 2 Introduction Transcription is a tightly regulated process in eukaryotic cells, which depends on the spatial and transient formation of non-covalent protein complexes and modulation of post-translational modifications (PTMs) to facilitate the organization of the transcription machinery {Schreiber, 2002 #145;Bernstein, 2007 #146}. Bromo and extra-terminal (BET) proteins provide a recruitment platform that helps aggregate large transcriptional complexes on active chromatin. They accomplish this by mediating interactions via their evolutionarily conserved bromodomain (BRD) modules, which recognize acetylated lysine (Kac) {Dhalluin, 1999 #100;Owen, 2000 #102;Jacobson, 2000 #111}. BETs, like all of the 42 human BRD-containing proteins, are also characterized by the presence of additional modular protein interaction domains that can help recruit other proteins to the acetylated protein target, forming complex assemblies that contribute to processes such as chromatin remodelling and transcription {reviewed in \Fujisawa, 2017 #112}. The BET sub-family comprises four proteins in humans (BRD2, BRD3, BRD4 and the testis-specific BRDT) that harbour at their amino-termini BRD modules with distinct specificity for Kac found on histones as well as a growing list of non-histone targets {reviewed in \Fujisawa, 2017 #112}, followed by an extra-terminal (ET) recruitment domain that mediates protein-protein interactions {Rahman, 2011 #37;Zhang, 2016 #127;Konuma, 2017 #136;Wai, 2018 #159} (Figure 1A). BRD4 and BRDT also contain a C-terminal motif (CTM) that binds to and facilitates the recruitment to acetylated chromatin of transcriptional regulators, including the positive transcription elongation factor b (P- TEFb), which phosphorylates RNA polymerase II (RNAPII; Figure 1B). BETs, and in particular BRD4, have been implicated in human disease, especially cancer {reviewed in \Fujisawa, 2017 #112}. Translocations of BRD4 (and more rarely BRD3) to the NUTM1 (NUT midline carcinoma family member 1) gene cause NUT midline carcinoma, a rare but aggressive form of squamous cell carcinoma {French, 2004 #133}. Furthermore, BRD4 levels are up-regulated in a variety of tumours, leading to aberrant expression of anti-apoptotic, cell cycle progression-related, and growth- promoting genes, including the MYC oncogene {Mertz, 2011 #138;Delmore, 2011 #110;Zuber, 2011 #137} and other transcription factors such as ERG, c-Myb, E2F1 and NF-κB. 3 The importance of BET proteins in cancer, together with the recognition that BRD-Kac interactions are druggable, has made them attractive targets for pharmaceutical intervention {Filippakopoulos, 2010 #16;Nicodeme, 2010 #106}. Indeed, direct targeting of BET BRDs by small molecule compounds such as the high affinity and specificity thienodiazepine (+)-JQ1 (hereafter referred to as JQ1), enables their displacement from Kac (Figure 1B). JQ1 (but not an inactive enantiomer) also displays anticancer activity in cell culture models, patient-derived xenograft models