A45
Tumour tissue profiling for biomarker discovery and validation at the drug targeting level: kinase activity
Rob Ruijtenbeek, Liesbeth Houkes, Rik de Wijn, Adrienne van den Berg, Monique Mommersteeg, Piet Boender, Riet Hilhorst
PamGene Int. B.V.,s-Hertogenbosch, Netherlands
Background
A novel biomarker discovery strategy is presented, which is based on measuring kinase activities in tumour tissue extracts. Discovery of markers at this enzymatic level, i.e. at the biological level many of the new targeted drug therapies intervene, is different from many other strategies measuring DNA/RNA/protein abundance. This activity-based approach is enabled by dynamic peptide microarrays, comprising peptidic substrates for phosphorylation by protein kinases.
Method
Patient derived tissues of different tumour types (e.g. breast, lung, kidney and blood) were used for extraction of total protein (6 slices of 10 um were sufficient for 20-100 microarray analyses). Equal amounts of total protein were analyzed for kinase activity on dynamic PamChip peptide microarrays. Dependent on the tissue source, amounts ranged from 1 5 ug per microarray analysis. Peptide microarrays comprised 144 or 256 different peptides. A PamChip96 plate format was used, enabling 96 microarray incubations run simultaneously.
Results
We show here that from all tumour tissue types tested kinase activity profiles could be generated robustly. Control experiments showed signal dependence on protein extract concentration, ATP concentration and finally, modulation of the peptide phosphorylations by kinase inhibitor drugs. Different phosphorylation profiles were obtained when the non-tumour tissue was compared to tumour tissue derived from the same patient, showing higher activities in the latter. Furthermore, different profiles were obtained that could be correlated to different patient classes.
Conclusion
A new biomarker discovery platform is presented, which is based on detection of kinase activities in protein extracts from patient derived tumour tissues by monitoring multiple peptide phosphorylation reactions on a peptide microarray. Additionally, the effect of a kinase inhibitor drug can be assessed in the same samples. This is an example of a biomarker discovery platform acting at the biological i.e. enzymatic - level the new targeted (kinase) drugs are effective.
