Precision medicine focused drug development has over the past years sparked a multitude of novel approaches, including the testing of patient-derived tumor tissue cultures for modeling
individual patient response, and the use of various molecular pathology techniques for advanced tumor profiling. Well-established genomics methodologies e.g. panel sequencing, however, cannot directly assess cell signaling activity within the tumor cells, as this is defined by the phosphorylation status of cellular signaling pathway networks.

Here we present the development of a robust protein profiling strategy utilizing the DigiWest assay platform, to
obtain data on the status of key cellular signaling networks implicated in cancer, and on proteins targeted by FDA-approved drugs including a number of targeted cancer therapies for EGFR, HER2, PI3K, mTOR, BCR-ABL, ALK and AKT. We compiled a list of important pathway nodes and their associated phosphorylation
sites that yield activity information on RAS/RAF/ERK, PI3K/AKT and mTOR signaling pathways. Based on this, we validated 242 total and phospho antibodies in a pre-set oncoproteomic DigiWest panel that yields information on the activity of these signaling networks from the receptor level down to transcription factors, apoptosis and proliferation.

We applied DigiWest panel profiling for elucidating drug response in pre-clinical cell models, in patient-derived 3d organoid models (PD3Ds, PDOs), and in clinical tumor samples. For example, we show differential effects of PI3K kinase inhibitor copanlisib vs
MEK inhibitor trametinib at different stages within their signaling networks in a cell-based model. Also, we performed oncoproteomic panel profiling in PD3D organoids that were subjected to phenotypic growth assays to assess their response to
common targeted therapies. In all cases, DigiWest multiplex profiling contributed valuable mechanistic insights to the phenotypic assays, thus representing an attractive platform for lead compound characterization in drug discovery and development.