Abstract Background: Acute myeloid leukemia (AML) is a malignant hematopoietic stem cell disease, caused by a differentiation block at the level of immature progenitors, leading to massive accumulation of malignant cells in the bone marrow and suppression of normal hematopoiesis. The disease is highly heterogeneous with 2 year survival rates, after intensive chemotherapy regimens depending on cytogenetic and molecular risk factors, varying between 70% for patients with good risk and less than 10% in those with poor prognostic features. In recent years clinical success has been achieved in the treatment of several cancer types with kinase inhibitors (KIs) and they may prove to be a valuable addition to standard therapy in AML. However, a proper rationale to select a suitable KI is still lacking. Phosphoproteomics based on tandem mass spectrometry (MS/MS) is a powerful approach for unbiased, global profiling of protein phosphorylation, and could identify aberrancies in signal transduction pathways and associated key kinases that may be suitable for targeted treatment with KIs. The aim of this study is to identify candidate driver kinases and signaling routes that may be targets for kinase inhibitor treatment by analyzing 16 selected AML cell lines. Methods: Sixteen different AML cell lines were cultured (EOL-1, HEL, HL-60, Kasumi-1, Kasumi-3, Kasumi-6, KG-1, KG-1a, ME-1, ML2, MM6, MOLM-13, MV-4-11, NB-4, OCI-AML3, THP1). For each cell line 10 mg protein lysate was processed. Phospho-Tyrosine containing peptides were captured from AML cell line lysates using the pY1000 antibody in an immunoprecipitation (IP) experiment. Both whole cell lysate and pTyr IP fractions were measured using nanoLC-MS/MS (2-hour gradient, QExactive). MaxQuant version 1.4.1.2 was used for phosphopeptide identification and label-free quantification. Results: The whole phosphoproteome dataset of 16 AML cell lines contained 4853 identified phosphopeptides corresponding to 2280 phosphoproteins, including 138 phosphokinases. Ranking of phosphokinases identified in each of the 16 AML cell lines confirmed FLT3, PDGFRa, JAK2 and c-KIT as hyperphosphorylated kinases in AML cells with mutations in these genes. Interestingly, AML cell lines without known kinase-related genomic aberrations also showed high activation of several kinases which could be potential KI targets. Current data analysis will link phosphokinases to downstream phosphoprotein substrates and pathways, in order to prioritize (combinations of) candidate driver kinases for functional perturbation experiments. Conclusion: Phosphoproteomics analysis of a panel of 16 AML cell lines showed differential (hyper-) activation of multiple kinases, including those already implicated in AML. The current analysis gives us insight into the heterogeneity of activated signaling pathways in AML and allows for the identification of possible new targets for treatment of AML with KIs. Citation Format: Carolien van Alphen, Jacqueline Cloos, Sander R. Piersma, Jaco C. Knol, Thang V. Pham, Gert J. Ossenkoppele, Henk Verheul, Jeroen JWM Janssen, Connie R. Jimenez. Phosphoproteomics of a panel of AML cell lines reveals oncogenic signaling and candidate drivers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1820. doi:10.1158/1538-7445.AM2015-1820