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

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.

Keywords: cancer metabolism; desmoplasia; fibrosis; inflammation; kinase inhibitors; kinase signatures; kinomic networks; pancreatic cancer; peptide array; transcription factors.

Figure A1

Differential phosphorylation levels of peptide sequences attributed to kinase family activity in PANC1 vs. wild-type control. Each red dot in a column represents one peptide sequence whose phosphorylation is performed by that column’s kinase family. The y axis reports log2-fold change and the x axis identifies the kinase family. Black dots represent peptides that did not demonstrate differential phosphorylation in PANC1 samples compared to wild-type samples, with the dashed horizontal line representing a positive or negative 0.2 log2-fold change cutoff. Red dots above these lines represent peptides that are more phosphorylated in PANC1 compared to control. Red dots that are below these lines represent peptides that are less phosphorylated in PANC1 compared to control.

Figure A2

Differential phosphorylation levels of peptide sequences attributed to kinase family activity in PDCL15 vs. wild-type control. Each red dot in a column represents one peptide sequence whose phosphorylation is performed by that column’s kinase family. The y axis reports log2-fold change and the x axis identifies the kinase family. Black dots represent peptides that did not demonstrate differential phosphorylation in PDCL15 samples compared to wild-type samples, with the dashed horizontal line representing a positive or negative 0.2 log2-fold change cutoff. Red dots above these lines represent peptides that are more phosphorylated in PDCL15 compared to control. Red dots that are below these lines represent peptides that are less phosphorylated in PDCL15 compared to control.

Figure A3

Differential phosphorylation levels of peptide sequences attributed to kinase family activity in PDCL5 vs. wild-type control. Each red dot in a column represents one peptide sequence whose phosphorylation is performed by that column’s kinase family. The y axis reports log2-fold change and the x axis identifies the kinase family. Black dots represent peptides that did not demonstrate differential phosphorylation in PDCL5 samples compared to wild-type samples, with the dashed horizontal line representing a positive or negative 0.2 log2-fold change cutoff. Red dots above these lines represent peptides that are more phosphorylated in PDCL5 compared to control. Red dots that are below these lines represent peptides that are less phosphorylated in PDCL5 compared to control.

Figure A4

Comparison of protein tyrosine kinases identified in patient-derived cell lines. For each comparison yellow circles represent differentially active protein tyrosine kinases in PDCL15, blue circles represent differentially active protein tyrosine kinases in PDCL5, and green overlapping area represents differentially active protein tyrosine kinases in PDCL15 and PDCL5. (A) Comparison of top 10 differentially active protein tyrosine kinases according to UKA and KRSA average percentile rankings. (B) Comparison of top 10 differentially active protein tyrosine kinases according to UKA and KRSA weighted average percentile rankings. (C) Comparison of top 10 differentially active protein tyrosine kinases according to all pipelines (KRSA, UKA, PTM-SEA, and KEA3) average percentile rankings. (D) Comparison of top 10 differentially active protein tyrosine kinases according to all pipelines (KRSA, UKA, PTM-SEA, and KEA3) weighted average percentile rankings.

Figure 1

Experimental design. (A) Patient-derived pancreatic cancer cells (light red) and wild-type pancreatic tissue specimens (yellow) are processed and diluted to a uniform protein concentration. (B) Samples are added to the PamChip array containing 196 consensus phosphopeptide sequences immobilized on porous ceramic membranes; two (purple and orange) such sequences are illustrated here. (C) Quantification of peptide phosphorylation levels. (D) Peptide phosphorylation data are analyzed with each of four independent bioinformatic pipelines (KRSA, UKA, PTM-SEA, KEA3) and then combined to generate a list of tyrosine protein kinases’ targets.

Figure 2

Outputs from upstream kinase identification pipelines for the commercially available PANC1 PDAC cell line compared to patient-derived wild-type pancreatic tissue. (A) Kinome Random Sampling Analyzer (KRSA); (B) Post-Translational Modification Signature Enrichment Analysis (PTM-SEA); (C) Kinase Enrichment Analysis Version 3 (KEA3); (D) Upstream Kinase Analysis (UKA); (E) Quartile summary. A more detailed figure legend can be found in Appendix B.

Figure 3

Outputs from upstream kinase identification pipelines for the patient-derived PDCL15 PDAC cell line compared to patient-derived wild-type pancreatic tissue. (A) Kinome Random Sampling Analyzer (KRSA); (B) Post-Translational Modification Signature Enrichment Analysis (PTM-SEA); (C) Kinase Enrichment Analysis Version 3 (KEA3); (D) Upstream Kinase Analysis (UKA). (E) Quartile summary. A more detailed figure legend can be found in Appendix B.

Figure 4

Outputs from upstream kinase identification pipelines for the patient-derived PDCL5 PDAC cell line compared to patient-derived wild-type pancreatic tissue. (A) Kinome Random Sampling Analyzer (KRSA); (B) Post-Translational Modification Signature Enrichment Analysis (PTM-SEA); (C) Kinase Enrichment Analysis Version 3 (KEA3); (D) Upstream Kinase Analysis (UKA). (E) Quartile summary. A more detailed figure legend can be found in Appendix B.

Figure 5

Summary figure illustrating kinases showing increased enzymatic phosphorylation activity in PDAC and their potential roles in the disease process. Solid black arrows indicate relationships between kinases or other proteins. (A) The neoteric kinase group includes candidate kinases potentially contributing to PDAC pathology in new or previously understudied ways; the reference kinase group includes kinases with well-established roles in human cancer pathophysiology. (B) Kinases are clustered by linkage to the processes that might underlie their involvement in PDAC.