Kim, Min-Sik

Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to conventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in >70% of patients. In summary, we present a comprehensive landscape of tyrosine phosphoproteome with EphB4 as a promising therapeutic target in pancreatic ductal adenocarcinoma.

Abstract Understanding the molecular profile of every human cell type is essential for understanding its role in normal physiology and disease. Technological advancements in DNA sequencing, mass spectrometry, and computational methods allow us to carry out multiomics analyses although such approaches are not routine yet. Human umbilical vein endothelial cells (HUVECs) are a widely used model system to study pathological and physiological processes associated with the cardiovascular system. In this study, next‐generation sequencing and high‐resolution mass spectrometry to profile the transcriptome and proteome of primary HUVECs is employed. Analysis of 145 million paired‐end reads from next‐generation sequencing confirmed expression of 12 186 protein‐coding genes (FPKM ≥0.1), 439 novel long non‐coding RNAs, and revealed 6089 novel isoforms that were not annotated in GENCODE. Proteomics analysis identifies 6477 proteins including confirmation of N ‐termini for 1091 proteins, isoforms for 149 proteins, and 1034 phosphosites. A database search to specifically identify other post‐translational modifications provide evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, and mono‐, di‐ and tri‐methylation events. Evidence for 11 “missing proteins,” which are proteins for which there was insufficient or no protein level evidence, is provided. Peptides supporting missing protein and novel events are validated by comparison of MS/MS fragmentation patterns with synthetic peptides. Finally, 245 variant peptides derived from 207 expressed proteins in addition to alternate translational start sites for seven proteins and evidence for novel proteoforms for five proteins resulting from alternative splicing are identified. Overall, it is believed that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.