Phosphotyrosine-based Phosphoproteomics for Target Identification and Drug Response Prediction in AML Cell Lines

• Published in: Molecular & cellular proteomics Vol. 19; no. 5; pp. 884 - 899
• Main Authors: van Alphen, Carolien, Cloos, Jacqueline, Beekhof, Robin, Cucchi, David G.J., Piersma, Sander R., Knol, Jaco C., Henneman, Alex A., Pham, Thang V., van Meerloo, Johan, Ossenkoppele, Gert J., Verheul, Henk M.W., Janssen, Jeroen J.W.M., Jimenez, Connie R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2020; The American Society for Biochemistry and Molecular Biology
• Subjects: acute myeloid leukemia; Antineoplastic Agents - pharmacology; Antineoplastic Agents - therapeutic use; Cell Line, Tumor; cell signaling; Cluster Analysis; Computer Simulation; drug targets; fms-Like Tyrosine Kinase 3 - metabolism; Humans; kinase inhibitors; Leukemia, Myeloid, Acute - drug therapy; Leukemia, Myeloid, Acute - genetics; Leukemia, Myeloid, Acute - metabolism; Leukemia, Myeloid, Acute - pathology; MAP Kinase Signaling System; mass spectrometry; molecular biology; Molecular Targeted Therapy; Mutation - genetics; personalized medicine; phosphoproteome; phosphoproteomics; Phosphorylation - drug effects; Phosphotyrosine - metabolism; Protein Kinases - metabolism; Proteome - metabolism; Proteomics; receptor tyrosine kinases; Reproducibility of Results; Tyrosine kinases; molecular biology; phosphoproteomics; kinase inhibitors; receptor tyrosine kinases; acute myeloid leukemia; cell signaling; Tyrosine kinases; drug targets; mass spectrometry; personalized medicine; phosphoproteome
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.RA119.001504

Phosphotyrosine (pY)-specific phosphoproteome profiles have been determined for 16 AML cell lines through a pY immunoprecipitation-based protocol. Kinase activity inference using dedicated ranking analysis, combining kinome-, activation loop-, and substrate-based analyses, identified potential drivers for all cell lines and driver function was confirmed through drug experiments. Results for two patient samples show potential of phosphoproteomics in a clinical setting. [Display omitted] Highlights •pY phosphoproteomes and dedicated ranking analyses for 16 AML cell lines.•RTK drivers, 6 mutant cell lines confirmed, identification for 4 more cell lines.•MAPK1/3 phosphorylation for cell lines without TK driver, indicating RAS mutation.•Drug target space phosphorylation correlates with drug IC50s in specific cell lines. Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal. In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidates for KI treatment, and efficacy of selected KIs was tested. Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation. Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.