Paradoxical MAPK-activation in response to treatment with tyrosine kinase inhibitors in CML: flow cytometry loses track

Paradoxical activation of the MAP-kinases, ERK1, and ERK2 (ERK1/2) is observed in CML cell lines and primary CML patient cells treated with tyrosine kinase inhibitors (TKI) in vitro. The commonly accepted assumption is that activated ERK1/2 is key regulators of survival of leukemic cells treated with kinase inhibitors. Hence, paradoxical ERK1/2-activation may trigger resistance in vivo, which yet has to be shown. We therefore sought to establish a flow cytometric assay that enables us to measure paradoxical TKI-induced ERK1/2-activation on a single cell basis in primary CML cells.Side-by-side Western blot and intracellular flow cytometry (FCM) after in vitro exposure of cell lines and primary cells to nilotinib were performed. Detailed analysis of pre-analytical factors and the issue of compartmentalization of phosphorylated ERK1/2 by confocal laser scanning microscopy were performed.Results were conflicting in that pERK-activation was robustly detected in Western blot assays, but not when cells were analyzed by FCM despite well functioning positive and negative controls. This is in contrast to experiments on other targets such as phospho-CrkL, where also in our hands TKI-dependent inhibition of phosphorylation is trackable by both Western blot and FCM assays.To our knowledge this is the first report of discordant results in phospho-protein analysis in TKI-treated cells analyzed by Western blot vs. FCM. We speculate that a substance specific interaction interferes with fluorescence dependent methods seeking to track phosphorylated ERK1/2 in TKI-treated cells.