Multiplexed Protein Analysis of Cellular Signal Transduction: Applications in Translational Research and Personalized Medicine

Abstract

Investigation of cellular signaling networks and their activation state is helpful for the study of diseases. Detailed proteomic analysis of signaling pathways can reveal how changes in protein expression patterns mediate the functional manifestation of disease-related (genetic) aberrations. A deepened understanding of proteomic alterations can help in the understanding and discovery of (novel) disease mechanisms for various conditions. Especially in the field of oncology, signaling proteins have become direct targets for drug intervention; thus, by providing accurate information on protein abundance and activation status, the analysis of cellular signaling presents a promising clinical tool for treatment evaluation in precision medicine. Likewise, in experimental clinical research, tangible information on individual proteins of interest and protein-mediated signaling as a whole can be used to evaluate action mechanisms of cell signaling-interfering drugs and aid to identify new target structures for future drug development. DigiWest is a novel, antibody-based targeted protein analytics method which combines the robustness and specificity of traditional Western Blot with the high-throughput nature of bead-based parallelized assay systems. In a single analysis, the method provides information on 200 or more signaling proteins and/or their activation state. High flexibility in analyte selection makes it highly adaptable for various research questions. Here, DigiWest was applied to evaluate efficacy and selectivity of tyrosine kinase inhibitors (TKI) as anti-cancer drugs in preclinical cellular models of FGFR2-fusion positive cholangiocarcinoma (iCCA). In a bedside to bench approach, analysis of signaling pathway modulation in treated cells carrying tumor driving FGFR2 fusions and resistance mutations was used to compare action mechanisms of different TKI. While FGFR2-selective and non-selective TKI showed similar effectiveness in treatment-sensitive cells, the non-selective TKI Lenvatinib proved superior in the presence of resistance mutations as was indicated by a conserved inhibition of key FGFR-related signaling pathways. In a precision medicine approach, DigiWest was employed to define personalized protein profiles in gastrointestinal tumors and identify individualized treatment options. A retrospective DigiWest analysis of pancreatic and colorectal tumors, enabled tumor stratification into subgroups within their respective entity based on pathway activity patterns, which were shown to match clinical patient characteristics. Methodological transfer of DigiWest into routine clinical setting was demonstrated through personalized characterization of 14 gastrointestinal tumors from patients presented to the molecular tumor board. Patient-specific protein/pathway activation profiles with drug-targetable alterations were revealed, which complemented genetic mutation analysis and provided additional information on treatment options. This highlights DigiWest as a suitable analytics tool for daily clinical precision oncology. In an application for basic molecular research, DigiWest was employed to uncover disease-specific signatures in patient-derived iPSC models of Schizophrenia. Analysis of protein expression profiles in differentiated neuronal cells (NPC) highlighted disease-induced changes to developmental pathways and demonstrated impaired differentiation capabilities on the protein level. These NPC stage-specific aberrations affected cell cycle control and DNA damage response and were correlated to p53 expression and phosphorylation. Here, the potential of DigiWest to uncover novel, protein-based disease-mechanistic insights was demonstrated. In this thesis, various applications of DigiWest were outlined. These evidently demonstrate the potential of this method for use in clinical, translational and basic research, e.g. in routine drug testing and disease mechanisms studies as well as in personalized medicine, specifically in clinical precision oncology settings.