Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Background. Whereas ALK translocations play a role in tumor growth in selected NSCLC cases, mutated forms of ALK have been shown to drive development of neuroblastoma, the most common malignant extracranial tumor of childhood. The mutation F1147L in the oncogene ALK increases the kinase activity, causing deregulated proliferation of sympatho-adrenal progenitor cells. We explored the use of kinase activity measurements to identify ALK activity in cell lysates and patient derived tissue lysates with the aim of identifying biomarkers to trace the activity of ALK in tumors and the effect of ALK inhibitors on these marker signals. Methods. Protein kinase activities were measured on PamChip® peptide microarrays comprising 144 Tyr containing peptide sequences from known human phosphorylation sites. The activity profiling experiments involved recombinant ALK kinase, lysates of cell lines overexpressing ALK and neuroblastoma tumors with elevated ALK F1174L presence. Per microarray analysis 5 ug of total protein was used in the presence of 100 uM ATP. Experiments were performed in the presence and absence of ALK kinase inhibitors crizotinib and NVP-TAE684. Real time kinetics of 144 peptide phosphorylations were measured with a fluorescently labeled antibody. Results. Phosphorylation profiles for neuroblastoma tumors, ALK overexpressing cell lines and recALK were generated. For recALK 48 novel peptide substrates were identified that showed signals depending on both ATP and kinase concentration and inhibition by the ALK inhibitors. In ALK overexpressing cell lines, but not in control cell lysates, signals on many ALK substrate peptides were significantly elevated, and returned to baseline level in the presence of ALK inhibitors. Whereas neuroblastomas with low ALK expression gave similar kinase activity profiles, the tumors harboring the ALK F1174L mutation were different, suggesting a more complex regulation of phosphorylation activity in these tumors. All tumors showed on-chip responses with ALK inhibitor drugs, but to different extent, which could be a basis for response prediction biomarker discovery. Conclusion. ALK activity of recombinant kinases, cell lysates and tumor lysates can be detected on PamChip® peptide microarrays. Based on results with model systems, this method shows promise to identify tumors that express elevated ALK activity and to study response of this class to ALK inhibitors.</jats:p>
<jats:p>Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1683. doi:1538-7445.AM2012-1683</jats:p>