Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases.

Justin F Creeden,Khaled Alganem,Robert E Mccullumsmith,Ali S Imami,Shi-He Liu,Tushar Tomar,F Charles Brunicardi,Rammohan Shukla,Faris Naji

doi:10.3390/ijms21228679

Abstract

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

Highlights

In vivo, kinases are heavily trafficked and compartmentalized to microdomains and cellular substructures where they act in concert with other kinases, receptors, and effector proteins to realize intricate signaling mechanisms that give rise to complex cellular behaviors
The signal intensities on the PamChip—which quantify the degree to which a peptide fragment has been phosphorylated—are lost when PamChip signatures are converted to the binary “differentially expressed” input categories required for Post-Translational Modification Signature Enrichment Analysis (PTM-SEA) or Kinase Enrichment Analysis Version 3 (KEA3) pipelines
This study presents evidence in support of the continued development of previously established inhibitory therapeutics that target select protein kinases, such as epidermal growth factor receptor (EGFR) [25], ephrin receptor A2 (EPHA2) [29], and SRC proto-oncogene kinase (SRC) [27], and our experimental results identify new pancreatic ductal adenocarcinoma (PDAC) targets, such as B lymphoid kinase (BLK), lymphocyte cell-specific kinase (LCK), and ABL proto-oncogene 2 kinase (ABL2), which may play a critical role in cancer cell biochemistry or desmoplastic inflammatory cell behavior

Summary

Introduction

Kinases are heavily trafficked and compartmentalized to microdomains and cellular substructures where they act in concert with other kinases, receptors, and effector proteins to realize intricate signaling mechanisms that give rise to complex cellular behaviors. Traditional assays remove each kinase of interest from its interacting partners and the physiologic conditions in which it normally operates By breaking these networks into discrete components and examining individual kinases in isolation, researchers discover initiators of kinase phosphorylation activity and catalogue the peptide targets that are activated or deactivated by direct phosphorylation by a specific kinase. These purification techniques incentivize researchers to minimize the integrity of a kinase’s normal physiologic environment in favor of a clean, assayable sample. We combined PamGene multiplexed kinome activity array data with four bioinformatic pipelines to identify protein kinases responsible for the differential phosphorylation activity observed in patient-derived and commercial pancreatic ductal adenocarcinoma (PDAC) cell lines compared to patient-derived wild-type pancreatic tissue specimens (Figure 1)

Methods

Results

Discussion

Conclusion