Pharmaceutical biotechnology has been emerging as a defined, increasingly important area of science dedicated to the discovery and delivery of drugs and therapies for the treatment of various human diseases. In contrast to the advancement in pharmaceutical biotechnology, current drug discovery efforts are facing unprecedented challenges. Difficulties in identifying novel drug targets and developing effective and safe drugs are closely related to the complexity of the network of interacting human proteins. Protein-protein interactions mediate virtually all cellular processes. Therefore both identification and understanding of protein-protein interactions are essential to the process of deciphering disease mechanisms and developing treatments. Unfortunately, our current knowledge and understanding of the human interactome is largely incomplete. Most of the unknown protein-protein interactions are expected to be weak and/or transient, hence are not easily identified. These unknown or uncharacterized interactions could affect the efficacy and toxicity of drug candidates, contributing to the high rate of failure. In an attempt to facilitate the ongoing efforts in drug discovery, we describe herein a series of novel methods and their applications addressing the broad topic of protein-protein interactions. We have developed a highly efficient site-specific protein cross-linking technology mediated by the genetically incorporated non-canonical amino acid L-DOPA to facilitate the identification and characterization of weak protein-protein interactions. We also established a protocol to incorporate L-DOPA into proteins in mammalian cells to enable in vivo site-specific protein cross-kinking. We then applied the DOPA-mediated cross-linking methodology to design a protein probe which can potentially serve as a diagnostic tool or a modulator of protein-protein interactions in vivo. To deliver such engineered proteins or other bioanalytical reagents into single live cells, we established a laser-assisted cellular nano-surgery protocol which would enable detailed observations of cell-to-cell variability and communication. Finally we investigated a possible experimental scheme to genetically evolve a fluorescent peptide, which has tremendous potential as a tool in cellular imaging and dynamic observation of protein-protein interactions in vivo. We aim to contribute to the discovery and development of new drugs and eventually to the overall health of our society by adding the technology above to the array of currently available bioanalytical tools. / text

Das normale Kolonepithel weist eine strenge Zellhierarchie auf, die aus bekannten Zelltypen besteht. Bei Darmkrebs (CRC) ist die Struktur weniger konserviert und nicht gut verstanden. Krebsauslösende Mutationen können die Prävalenz von Zelltypen verändern, und Zellen können sich auch dedifferenzieren, um einer gezielten Krebstherapie zu entgehen. Mein Ziel ist es, die Existenz heterogener Zelltypen in Organoiden zu bestätigen und Signalnetzwerke in CRC zu untersuchen, indem ich mit pharmakologischen Eingriffen spezifische Signalwege inhibiere, die Zellhierarchien im normalen Darm kontrollieren. Strategisch ausgewählte Medikamente wurden eingesetzt, um Knotenpunkte in verschiedenen Signalwegen zu hemmen, die für das Fortschreiten von Darmkrebs relevant sind. Ich untersuchte, ob die Inhibition von Signalwegen die Zusammensetzung der Zelltypen und den Differenzierungszustand verändert oder welche Kombinationen von Inhibitoren Plastizität oder Apoptose auslösen könnten. Von Patienten stammende Organoide mit verschiedenen onkogenen Treibermutationen wurden kultiviert und 48 Stunden lang mit einer Reihe von Inhibitoren und Inhibitorkombinationen behandelt. Diese Organoide wurden hauptsächlich auf zwei Ebenen untersucht: durch scRNA seq zur Ermittlung ihres Transkriptoms und durch CyTOF, das die Proteinhäufigkeit pro Zelle misst, um die Aktivität von Signaltransduktionskaskaden zu beurteilen. Beide Methoden wurden eingesetzt, um die Heterogenität des CRC zu quantifizieren. Ich konnte feststellen, dass sich Organoide mit denselben Treibermutationen ähnlicher verhalten und dass die molekularen Grundlagen der verschiedenen Linien Unterschiede im Therapieerfolg bedingen. Heterogene Transkriptome und Proteinexpression wurden durch einen Differenzierungsgradienten beeinflusst und konnten durch die Zugabe von Inhibitoren verändert werden. Die MAPK-Aktivität folgt diesem Differenzierungsgradienten und eine MAPK-Inhibition verringerte die Zellheterogenität und führte zu Plastizität. Darüber hinaus stellte ich fest, dass ein Teil der Zellen in Apoptose geht und die verbleibenden Zellen einen nicht-proliferativen Stammzellzustand annehmen, der es den Zellen ermöglicht, sich nach Aussetzung der Behandlung zu erholen. Es wurden in silico und in vitro Analysen durchgeführt, um neuartige Inhibitorkombinationen zur Maximierung der Apoptose in CRC-Organoiden zu finden, um die Entstehung therapieresistenter Subpopulationen weiter zu reduzieren. Wirksame Behandlungskombinationen bleiben jedoch zelllinienabhängig. Durch die getrennte Analyse des Zelldifferenzierungszustands und des Zellsignalisierungszustands habe ich dazu beigetragen zu verstehen, wie Tumorzellen einer gezielten Therapie durch nicht-genetische Resistenzmechanismen entgehen können. Die MAPK-Inhibition zur Verringerung der Zellheterogenität in Kombination mit anderen Inhibitoren könnte in Zukunft zur Optimierung des Therapieerfolgs eingesetzt werden. / Normal colon epithelium has a strict cell hierarchy consisting of well-known cell types. In colorectal cancer (CRC) the structure is less conserved and poorly understood. Cancer driver mutations may modulate the prevalence of cell types, and cells may also dedifferentiate to overcome targeted cancer therapy. My aim is to confirm the existence of heterogeneous cell types in organoids and investigate signaling networks in CRC by targeting specific signaling pathways with pharmacological intervention, which control cell hierarchies in the normal intestine. Strategically selected drugs were used to inhibit nodes in different signaling pathways relevant to the progression of CRC. I explored whether signaling inhibition changes cell type composition and differentiation state, or which inhibitor combinations might induce plasticity or apoptosis. Patient-derived organoids with different oncogenic diver mutations were cultured and treated with a panel of inhibitors and inhibitor combinations for 48 hours. These organoids were mainly examined on two levels: by scRNA seq to assess their transcriptome and by CyTOF, which measures protein abundance to assess the activity of pathways. Both methods were used to quantify CRC heterogeneity. I was able to see that organoids with the same driver mutations behave more similarly and that the molecular underpinnings of the different lines drive differences in therapy response. Heterogeneous transcriptomes and protein expression were affected by a differentiation gradient and could be altered by inhibitor addition. MAPK activity was graded along this differentiation gradient, and MAPK inhibition reduced cell heterogeneity and induced plasticity. Additionally, I found that a fraction of cells undergo apoptosis, and the remaining cells adopt a non-proliferative stem cell state, which allows cells to recover after suspension of treatment. \textit{In silico} and \textit{in vitro} analyses were performed to find novel inhibitor combinations to maximize apoptosis in CRC organoids to further reduce the emergence of therapy-resistant subpopulations. However, effective treatment combinations remain cell-line dependent. By separately analyzing cell differentiation state and cell signaling state I contributed to our understanding of how tumor cells can evade targeted therapy by non-genetic resistance mechanisms. Using MAPK inhibition to reduce cell heterogeneity in combination with other inhibitors may be used in the future to optimize therapy success.