Inference of Kinase-Signaling Networks in Human Myeloid Cell Line Models by Phosphoproteomics using Kinase Activity Enrichment Analysis (KAEA)

Despite the indisputable efficacy of kinase-inhibitors in myeloid malignancies, cure remains an exception and most patients eventually progress. Therefore, there is an unmet clinical need in developing diagnostic pipelines for the characterization of kinase-activities and signaling networks to investigate their implications in response and resistance. Here, we used the BCR-ABL1 driven K562 and hetero-/homozygote FLT3-ITD driven MOLM13/MV4-11 human myeloid cell line models exposed to the clinically established BCR-ABL1 and FLT3 kinase-inhibitors, Nilotinib and Midostaurin, respectively. Titanium dioxide enrichment with liquid chromatography tandem mass spectrometry (LC-MS) was used and the differential phosphoproteomics profiles analyzed with the Kinase-Activity Enrichment Analysis (KAEA) pipeline. This novel pipeline allows inferring kinase activities within signaling networks. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib as well as the indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. With our pipeline, we provide researchers and clinicians an instrument to monitor biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted and ongoing to determine the utility of our pipeline to characterize mechanisms of disease progression and treatment failure.