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Tyrosine Phosphoproteomics of Patient-Derived Xenografts Reveals Ephrin Type-B Receptor 4 Tyrosine Kinase As a Therapeutic Target in Pancreatic Cancer

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Tyrosine Phosphoproteomics of Patient-Derived Xenografts Reveals Ephrin Type-B Receptor 4 Tyrosine Kinase As a Therapeutic Target in Pancreatic Cancer cancers Article Tyrosine Phosphoproteomics of Patient-Derived Xenografts Reveals Ephrin Type-B Receptor 4 Tyrosine Kinase as a Therapeutic Target in Pancreatic Cancer Santosh Renuse 1,2 , Vijay S. Madamsetty 3 , Dong-Gi Mun 1, Anil K. Madugundu 1,4,5, Smrita Singh 1,4,5 , Savita Udainiya 1,6, Kiran K. Mangalaparthi 1,4,7, Min-Sik Kim 8,9, Ren Liu 10, S. Ram Kumar 11, Valery Krasnoperov 12, Mark Truty 13, Rondell P. Graham 1, Parkash S. Gill 10, Debabrata Mukhopadhyay 3,* and Akhilesh Pandey 1,2,5,6,* 1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA; [email protected] (S.R.); [email protected] (D.-G.M.); [email protected] (A.K.M.); [email protected] (S.S.); [email protected] (S.U.); [email protected] (K.K.M.); [email protected] (R.P.G.) 2 Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA 3 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Jacksonville, FL 32224, USA; [email protected] 4 Institute of Bioinformatics, International Technology Park, Bangalore 560066, Karnataka, India 5 Manipal Academy of Higher Education (MAHE), Manipal 576104, Karnataka, India Citation: Renuse, S.; Madamsetty, 6 Center for Molecular Medicine, NIMHANS, Bangalore 560027, Karnataka, India 7 V.S.; Mun, D.-G.; Madugundu, A.K.; Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham University, Kollam 69025, Kerala, India 8 Singh, S.; Udainiya, S.; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Mangalaparthi, K.K.; Kim, M.-S.; Liu, Baltimore, MD 21205, USA; [email protected] 9 Department of Biological Chemistry, Johns Hopkins University School of Medicine, R.; Kumar, S.R.; et al. Tyrosine Baltimore, MD 21205, USA Phosphoproteomics of Patient- 10 Departments of Pathology and Medicine, Keck School of Medicine of the University of Southern California, Derived Xenografts Reveals Ephrin Los Angeles, CA 90033, USA; [email protected] (R.L.); [email protected] (P.S.G.) Type-B Receptor 4 Tyrosine Kinase as 11 Departments of Surgery, Keck School of Medicine of the University of Southern California, a Therapeutic Target in Pancreatic Los Angeles, CA 90033, USA; [email protected] Cancer. Cancers 2021, 13, 3404. 12 VasGene Therapeutics Inc., Los Angeles, CA 90033, USA; [email protected] https://doi.org/10.3390/cancers 13 Division of Hepatobiliary and Pancreas Surgery, Mayo Clinic, Rochester, MN 55905, USA; 13143404 [email protected] * Correspondence: [email protected] (D.M.); [email protected] (A.P.); Tel.: +1-904-953-6177 (D.M.); +1-507-293-9564 (A.P.) Academic Editors: Udayan Guha, Xu Zhang and Maxim V. Berezovski Simple Summary: Pancreatic cancer is one of the deadliest solid malignancies. Pancreatic ductal Received: 22 March 2021 adenocarcinoma accounts for 90% of pancreatic cancer cases with minimal response to traditional Accepted: 28 June 2021 chemotherapies. Protein tyrosine kinases have been shown to be hyperactivated in cancers and thus Published: 7 July 2021 can serve as therapeutic targets. Patient-derived tumor xenografts (PDXs) in animal models such as mice are an appropriate resource to identify such activated kinases. PDXs models are excellent for the Publisher’s Note: MDPI stays neutral identification of therapeutic targets as compared to cell line models as they better reflect an in vivo with regard to jurisdictional claims in environment. We identified ephrin type-B receptor 4 (EphB4) as hyperactivated in PDXs derived published maps and institutional affil- from pancreatic ductal adenocarcinoma. iations. Abstract: Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to con- ventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the Copyright: © 2021 by the authors. discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic Licensee MDPI, Basel, Switzerland. strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts This article is an open access article (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and distributed under the terms and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass conditions of the Creative Commons spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to Attribution (CC BY) license (https:// profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This creativecommons.org/licenses/by/ 4.0/). analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. Cancers 2021, 13, 3404. https://doi.org/10.3390/cancers13143404 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3404 2 of 16 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 tis- sue 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. Keywords: EphB4; receptor tyrosine kinase; targeted therapy; personalized medicine 1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer in the USA and is predicted to be the second leading cause by the year 2030 [1,2]. The overall 5-year survival rate of PDAC patients from all stages of the disease combined is less than 10% and has not increased in the past five decades [3]. There are no effective therapies for PDAC that can cure or substantially increase survival, and only about 10%–20% of patients are considered candidates for surgery as most patients present with metastatic cancer. Gemcitabine chemotherapy in combination with the EGFR inhibitor erlotinib showed modest improvements in the overall survival rate [4]. Thus, characterization of the underlying molecular mechanisms responsible for the development of PDAC may lead to the identification of novel therapeutic targets. Several genetic alterations, including KRAS, CDKN2A, TP53, SMAD4, and BRCA2, have been implicated in the pathogenesis of PDAC [5,6]. Aberrant activation of kinase signaling pathways is commonly observed in several malignancies. Activating mutations in kinases such as EGFR represent a major mechanism of oncogenesis. Kinases regulate most cellular processes responsible for cell growth and dif- ferentiation through reversible phosphorylation. Thus, kinases have emerged as attractive candidates for targeted therapy in a number of cancers [7,8]. Phosphoproteomics offers a functional readout in biological systems reflecting proteins and pathways that are activated, thus acting as a surrogate to identify activated kinases. We and others have used this approach to characterize aberrant kinase signaling in various cancers and demonstrated its utility in identifying potential therapeutic targets [9–11]. Thus, the identification of oncogenic tyrosine kinases activated across a broad spectrum of PDACs is a promising strategy for the development of new targeted therapies and for individualizing treatment. Various kinases have been found to be implicated in PDAC development and progres- sion, such as EGFR, which is required for acinar-ductal metaplasia (ADM). Overexpression of EGFR and related receptor, ERBB3, has been detected in PDAC [12], whereas pseudoki- nase 223 (SgK223) has been shown to be increased by phosphorylation in PDAC cell line models through JAK1/Stat3 signaling [13]. Tao et al. studied proteome and phosphopro- teome of patient-derived PDAC tissues and identified several extracellular matrix-related proteins associated with PDAC [14]. Le Large et al. studied the phosphoproteome of 11 pancreatic cancer cell lines and identified focal adhesion kinase (FAK) as a potential synergistic target with Nab-paclitaxel [15]. Humphrey et al. carried out phosphotyrosine profiling of 36 PDAC cell lines and observed three different patterns based on the pro- files [16]. However, there is still a lack of studies describing phosphotyrosine signaling in PDAC tissues and PDX models. In this study, we carried out phosphotyrosine profiling of a number of PDXs to discover activated tyrosine kinase signaling pathways in PDAC. We employed antibody- based enrichment of phosphotyrosine peptides followed by a liquid chromatography-mass spectrometry-based label-free quantitation approach to identify and quantify phospho- tyrosine peptides among these PDXs. In all, we identified 1199 unique phosphorylation sites mapped to 704 proteins in one or more PDX samples. While there was considerable Cancers 2021, 13, 3404 3 of 16 heterogeneity in the phosphopeptides identified across individual cases, we identified several recurring activated tyrosine kinases such as the EPH receptor B4 (EphB4). We vali- dated EphB4 overexpression using pancreatic ductal adenocarcinoma tissue microarrays (TMAs) using immunohistochemistry (IHC). In vivo studies directed
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