Establishing stable cell lines for the generation of interaction profiles of proteins involved in RNA editing

Adenosin zu Inosin Editierung von Ribonukleinsäuren (RNA) ist eine konservierte post-transkriptionelle Modifikation in höheren Metazoen. Die hydrolytische Desaminierung von Adenosin zu Inosin wird von einer Enzymfamilie durchgeführt, welche als Adenosin-Deaminasen bekannt sind (ADARs) und doppelsträngige RNA desaminieren. Durch RNA-Editierung wird die Sequenz eines primären Transkriptes verändert, was dramatische Auswirkungen haben kann: Editierung der mRNA eines kodierenden Transkriptes kann zu alternativem Splicing und Aminosäurensubstitutionen im translatierten Protein führen. Abgesehen davon gehören nicht-codierende RNAs und repetitive Sequenzen, wie Alu-Elemente und micro RNAs, zu den Hauptsubstraten von ADARs. Editierung dieser Substrate kann Einfluss auf die Expression von Genen haben, weil die Sequenz regulatorischer Elemente und kleiner RNAs verändert wird. Durch aktuelle Studien wurden bereits viele Substrate für ADAR-vermittelte Editierung identifiziert. Jedoch sind Auswirkungen der Editierung auf die Funktion vieler prozessierter Substrate noch nicht bekannt. Zusätzlich zu den unbekannten Konsequenzen der RNA-Editierung gibt auch die Regulation dieses Mechanismus Rätsel auf. In den vergangenen Jahren wurden in unserer Arbeitsgruppe einige Kandidaten identifiziert, welche als mögliche Regulatoren für RNA-Editierung in Frage kommen. In diesem Projekt haben wir versucht, diese Kandidaten stabil in Säugerzellen zu exprimieren, um durch Aufreinigungsmethoden und anschließende massenspektrometrische Analyse Interaktionsnetzwerke dieser Kandidaten aufzuklären. Der zweite Teil dieser Arbeit behandelt zwei Substrate für RNA-Editierung: das zytoskelettale Protein Filamin A und BLCAP, ein Protein, assoziiert mit der Entstehung von Blasenkrebs. Editierte und nicht editierte Versionen dieser Proteine wurden stabil in Säugerzellen exprimiert. Durch Aufreinigungsexperimente unter nativen Bedingungen und anschließender massenspektrometrischer Analyse konnten einige Proteine identifiziert werden, welche mit diesen ADAR-Substraten interagieren.Adenosine to inosine RNA editing is a posttranscriptional modification highly conserved in higher metazoa. The hydrolytic deamination of adenosine to inosine is catalysed by a family of enzymes, known as adenosine deaminases that act on double-stranded RNA (ADARs). Changing the sequence of a primary transcript by RNA editing can have dramatic consequences: Editing of the pre-mRNA of a coding transcript can lead to alternative splicing events and may cause amino acid substitutions in the translated protein, as the triplet codon becomes changed. Apart from that, most of the known substrates of RNA editing are non-coding RNAs and repetitive elements, as Alu elements in untranslated regions of the transcript, and micro RNAs. These editing events can influence gene expression, as the sequence of regulatory elements or the target specificity of small RNAs is altered. To date, on-going studies have identified many targets of ADAR editing. However, very little is known about the consequences of the editing events. In addition to the consequences of editing on its targets, mechanisms of regulation of A to I editing are still unclear. In the last few years several candidates for regulators of ADAR activity have been identified in our lab. In this thesis we stably expressed these candidates in mammalian cell lines for purification assays. Subsequent mass spectrometric analysis of purified complexes led to the identification of proteins interacting with editing regulator candidates, what may help to clarify regulatory networks involved in A to I RNA editing. The second part of this project deals with two protein-coding targets of RNA editing: The effect of RNA editing on the interaction profiles of the cytoskeletal cross-linker Filamin A, and the bladder cancer associated protein BLCAP is investigated. Edited and unedited versions of both proteins were stably expressed in mammalian cell lines. Purification of the two targets of RNA editing under native conditions led to the identification of interacting proteins after mass spectrometric analysis

Preclinical Characterization of a Next-Generation Brain Permeable, Paradox Breaker BRAF Inhibitor

PURPOSE Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation