Histone acetylation is an epigenetic post-translational modification recognised by the bromodomain, a protein module that forms part of multi-component complexes affecting transcription. This interaction plays fundamental cellular roles, and shows association with particular diseases including inflammation and cancer. The biological roles of bromodomains and the progress of ligands developed so far has been summarised in introductory Chapter 1. Work within the group has led to the development of a nanomolar ligand for BRD4, a BET bromodomain implicated in cancer and numerous diseases. Evaluation in an NCI-60 cancer cell screen indicated antiproliferative activity in a variety of cancer types. However, metabolic predictions indicate that this compound is unoptimised for use in vivo. Chapter 2 describes synthesis of a collection of analogues to improve the physical and pharmacokinetic properties of this series of compounds. This work identified compounds with equivalent affinity but greater predicted metabolic stability, as well as more potent derivatives. This research will direct the design of potent and metabolically stable derivatives that can be used in animal models. Chapter 3 describes work carried out towards the development of small molecules to target bromodomains for which there are no known ligands, using the FALZ bromodomain as an initial target. A fragment-based approach has identified a number of compounds that bind to different non-BET bromodomains. These fragments will be a useful starting point for the development of more potent and selective non-BET bromodomain ligands. As well as acetylated lysines, a number of other acylation post-translational modifications occur on lysine residues. Chapter 4 describes work carried out to investigate the interaction of other acylated lysine residues with bromodomains. This work highlighted that other acylated lysines can interact with bromodomains, and selectivity for particular bromodomains can also be achieved. These modified lysines could be incorporated into cognate peptides to improve in vitro peptide displacement assays, aiding the development of small molecular bromodomain probes.

Medulloblastomas are the most frequent malignant childhood brain tumors, arising in the posterior fossa of children. The overall 5-year survival is 70%, although children often suffer severe long-term side effects from standard medical care. To improve progression-free survival and quality of life for these children, finding new therapeutic targets in medulloblastoma is imperative. Medulloblastoma is divided in to four molecular subgroups (WNT, SHH, Group 3 and Group 4) based on key developmental pathways essential for the initiation and maintenance of tumor development. The MYC family of proto-oncogenes regulates cell proliferation and differentiation in normal brain. Aberrant expression of MYC proteins occurs commonly in medulloblastoma. Our studies on Group 3 medulloblastoma identify the transcription factor SOX9 as a novel target for the E3 ubiquitin ligase FBW7, and show that increased stability of SOX9 confers an increased metastatic potential in medulloblastoma. Moreover, SOX9-positive cells drive distant recurrences in medulloblastoma when combining two regulatable TetON/OFF systems. MYCN depletion leads to increased SOX9 expression in Group 3 medulloblastoma cells, and the recurring tumor cells are more migratory in vitro and in vivo. Segueing to treatment of medulloblastoma, we show that BET bromodomain inhibition specifically targets MYC-amplified medulloblastoma cells by downregulating MYC and MYC-transcriptional targets, and that combining BET bromodomain- and cyclin-dependent kinase- inhibition improves survival in mice compared to single therapy. Combination treatment results in decreased MYC levels and increased apoptosis, and RNA-seq confirms upregulation of apoptotic markers along with downregulated MYC target genes in medulloblastoma cells. This thesis addresses novel findings in transcription factor biology, recurrence and treatment in Group 3 medulloblastoma, the most malignant subgroup of the disease.

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Bromodomains (BRDs) are protein modules which bind to acetylated lysines on histones and transcriptional regulating proteins. BRD-containing proteins are involved in a large variety of critical cellular processes and their misregulation, or mutation of the genes encoding for them, has been linked to pathogenesis in humans. The generation of chemical probes (potent, selective and cell permeable small molecules) in cellular experiments to investigate the biological role of the BRDs is thus desirable. A chemical probe for the CREB (cyclic-AMP response element binding protein) binding-protein (CBP) and E1A binding protein (p300) BRDs was developed, starting from a low molecular weight, weak and non-selective dimethylisoxazole benzimidazole compound. Parallel synthesis was used to optimise the initial hit into a weak, but selective CBP inhibitor. Further modification of the two N-1 and C-2 moieties of the benzimidazole scaffold, led to highly potent and selective CBP inhibitors. Structure-guided design was then applied to optimise the selectivity of the series for CBP over the first domain of bromodomain-containing protein 4 BRD4(1). A strategy to reduce the flexibility of the N-1 and C-2 ethylene linker groups through the incorporation of conformational constraints led to inhibitors with increased selectivity. The optimal compound was highly potent for the CBP and p300 BRDs (K_d 21 nM and 32 nM, respectively) and selective over BRD4(1) (40-fold and 27-fold, respectively). On-target cellular activity was observed in a fluorescence recovery after photobleaching (FRAP) assay (0.1 μM), a p53 reporter gene assay (IC₅₀ 1.5 μM) and a Förster resonance energy transfer (FRET) assay (5 μM). A weak indolizine bromodomain-containing protein 9 (BRD9) inhibitor was used as the starting point for the development of a BRD9/BRD7 chemical probe. Analogues were synthesised via [3+2] cycloadditions. An optimised compound was found to be highly potent (68 nM) and selective over BRD4(1) (34-fold). On-target cellular activity was observed in a FRAP assay (5 μM). Efforts were made to improve the cellular activity through the introduction of an ionisable centre to aid solubility. A selection of piperazine analogues were shown to be potent and selective, and these compounds warrant further investigation of their selectivity and cellular activity. Overall, the work has led to the first potent and selective inhibitors of the CBP/p300 and BRD9 BRDs. It also highlights the role of structural analysis in the development of inhibitors that modulate protein-protein interactions.

Les protéines à bromodomaines (BCPs) sont notamment impliquées dans la régulation de la transcription de gènes et la signalisation cellulaire. Leur dérégulation conduit au développement pathologies, telles que les maladies inflammatoires, cardiovasculaires et plus particulièrement les cancers. Les BCPs sont capables de reconnaitre les lysines acétylées de protéines histones via leur module BromoDomaine(s) (BDs). Parmi les huit familles de BCPs, mon projet de thèse s’intéresse à la famille « BET ». Celle-ci, comprend quatre protéines constituées de deux BDs en tandem, formant deux sous-familles BD1 et BD2. L’architecture de la cavité centrale des BDs, qualifiée de « druggable », a permis l’émergence de ces protéines en tant que nouvelles cibles épigénétiques prometteuses. À ce jour, une vingtaine d’essais cliniques ont été initiés pour des molécules « pan-BET », inhibant l’ensemble des membres de cette famille. Cependant, l'inhibition « pan-BET » est problématique au niveau clinique puisqu’elle impacte de nombreuses voies de transcription et engendre l’apparition de cellules résistantes. Mon projet de thèse s’intègre au challenge actuel qui est de développer des inhibiteurs plus sélectifs, par exemple envers l’une des sous-familles BD1 ou BD2 ou plus idéalement envers un seul BD de la famille BET. Le développement de « sondes épigénétiques sélectives » ciblant des BDs de la famille BET, devrait permettre de décrypter leur rôle et leur mécanisme d’action dans les divers processus biologiques. L’identification de « candidat médicament » devrait aboutir à de nouvelles thérapies ciblées et de pallier les résistances liées à l’utilisation de molécules pan-BET inhibitrices. / Bromodomain-containing proteins (BCPs) are especially involved in the regulation of gene transcription and cell signalling. Their dysregulation lead to the development of pathologies, such as inflammatory, cardiovascular diseases, and more particularly cancers. BCPs involved in the recognition of acetylated lysine of the histone tails, through their BromoDomain(s) module(s) (BDs). Among the eight families of BCPs, my thesis project focuses on the “BET” family. This family comprises four proteins which are composed of a tandem of two BDs each belonging to the BD1 or the BD2 subfamily. The architecture of the central cavity of the BDs, qualified as "druggable", allows the emergence of these proteins as new promising epigenetic targets. To date, about twenty clinical trials targeting different types of cancer have been initiated for "pan-BET" molecules that target all the members of this family. However, "pan-BET" inhibition is clinically problematic because it impacts many transcriptional pathways and causes the appearance of resistant cells. My thesis project is part of the current challenge is to develop more selective inhibitors, for example towards the BET-BD1 subfamily or the BET-BD2 subfamily or ideally towards one single BD inside the BET family. The development of such "selective epigenetic probes" targeting BET family BDs should allow deciphering their role and mechanism of action in various biological processes. Identifying "drug candidates" should lead to new targeted therapies and overcome the resistances related to the use of pan-BET molecules.

ATAD2 est un facteur très conservé mais peu caractérisé qui possède différents domaines fonctionnels : un domaine AAA ATPase et un bromodomaine (BRD). Normalement, ATAD2 est exprimé fortement dans les cellules germinales males ainsi que dans les cellules souches embryonnaires (cellules ES). De plus, la surexpression de cette protéine a été détectée dans de nombreux cancers. Il a été montré qu'ATAD2 agit comme co-activateur des récepteurs aux androgènes et aux œstrogènes. Cette protéine semble aussi agir comme co-facteur de l’oncogène Myc et joue un rôle dans la voie pRb/E2F. La surexpression d’ATAD2 prédit un mauvais prognostic dans les cancers du poumon et du sein. Toutes ces caractéristiques font d'ATAD2 un candidat de choix comme biomarqueur pronostic et une cible potentielle pour des agents thérapeutiques dans le cadre de cancers agressifs.Dans ce projet de thèse, nous montrons que hATAD2 interagit avec l'histone H4 acétylée via son bromodomaine, et que le domaine ATPase est responsable de la multimérisation d’ATAD2 et permet au BRD d’interagir avec les lysines acétylées dans les cellules. Des investigations complémentaires, comprenant notamment des études structurales, montrent que le BRD d'ATAD2 est responsable de son interaction spécifique avec la forme acétylée de la lysine 5 de l'histone H4. Nous avons aussi analysé le domaine AAA ATPase et découvert des éléments qui contrôlent son rôle dans la multimérisation des protéines. De plus, nous avons étudié ATAD2 dans la lignée de cellules cancéreuses pulmonaires, H1299, ainsi que dans les cellules ES et démontré que ce facteur est essentiel pour la prolifération des cellules en l'absence des facteurs de croissance. En combinant des approches ChIP-seq, ChIP-protéomics et RNA-seq dans les cellules ES, nous avons montré qu'ATAD2 est très enrichi dans les régions à haute activité transcriptionnelle et maintient la chromatine accessible pour les facteurs impliqués dans les activités de la chromatine. Ces données indiquent qu'ATAD2, dans son contexte physiologique, assure un rôle essentiel dans les activités générales de la chromatine, telles que la transcription, en maintenant l'accessibilité de la chromatine pour les facteurs de transcription.Enfin, afin de caractériser la structure d’ATAD2 et celle de son homologue dans Schizosaccharomyces pombe, ABOI, différents fragments contenants le domaine AAA ATPase ont été produits dans des bactéries ainsi que dans des cellules d'insectes en utilisant des vecteurs d’expression de baculovirus. Les conditions de production de fragments solubles ont été établies et certains de ces fragments ont été purifiés. Néanmoins, l’obtention de la structure cristalline de l'ATAD2 nécessite des travaux supplémentaires. / ATAD2 is an evolutionarily conserved but poorly characterized factor that bears different types of func¬tional domains: an AAA ATPase domain and a bromodomain (BRD). ATAD2 is normally highly ex¬pressed in male germ cells and in embryonic stem cells (ESC), however the overexpression of this protein has been detected in a large variety of independent cancers. ATAD2 is proposed to act as a co-activator of androgen and estrogen receptors and in addition, this protein also seems to act as a co-factor for Myc oncogene and plays a role in the pRb/E2F pathway. Moreover, the overexpression of ATAD2 predicts poor prognosis in lung and breast cancers. All of these characteristics make ATAD2 a valuable prognosis biomarker and a promising therapeutic target in aggressive cancers.Herein, we show that hATAD2 binds to acetylated H4 tail through its BRD, and that its ATPase domain enables ATAD2 multimerization, affecting the ability of the BRD to bind acetylated lysine in cells. Additional investigations, including structural studies, show that ATAD2’s BRD is responsible for its specific interaction with acetylated lysine 5 of histone H4. We have also functionally analyzed the AAA ATPase domain and discovered elements that control its role in protein multimerization. In addition, we studied ATAD2 in ESC and in the H1299 lung cancer cell line, and demonstrated that this factor has crucial roles in cell proliferation in the absence of growth factors. Moreover, by using a combination of ChIP-seq, ChIP-proteomics and RNA-seq experiments in ESC, we found that ATAD2 is highly enriched in regions with high transcriptional activity and that it keeps chromatin accessible for chromatin templated factors. These data indicate that ATAD2 in its physiological context ensures a critical role in general chromatin-templated activities, such as transcription, by maintaining the accessibility of chromatin for transcription factors. Finally, in order to structurally characterize either ATAD2 or its homologue in Schizosaccharomyces pombe, ABOI, different fragments containing the AAA ATPase domain were produced in bacteria as well as in insect cells using baculovirus expression vectors. Conditions to produce soluble fragments were established and some of these fragments were purified. Nonetheless, solving the crystal structure of ATAD2 still requires further investigation.