Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3’s substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment.
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We thank Dr. W. Andy Tao (Department of Biochemistry, Purdue University) for his helpful discussions and insights in adapting the kinase assay linked with phosphoproteomics technique. We thank the University of Minnesota’s Center for Mass Spectrometry and Proteomics for assistance in data acquisition and analysis. We thank Dr. Anton B. Iliuk (Tymora Analytica, West Lafayette, IN) for his support and insight in implementing the PolyMAC phosphopeptide enrichment kit. * This work was supported by the National Cancer Institute, Innovative Molecular Analysis Technologies (IMAT), and the Center to Reduce Cancer Health Disparities Diversity Training Branch: R01CA182543, R33CA183671, and R01CA182543-S1.
* This work was supported by the National Cancer Institute, Innovative Molecular Analysis Technologies (IMAT), and the Center to Reduce Cancer Health Disparities Diversity Training Branch: R01CA182543, R33CA183671, and R01CA182543-S1. □S This article contains supplemental material.