Fig. 5

Analysis of FLT3 resistance via pHASED. A Number of differentially phosphorylated peptides (log₂ ± 0.5, p ≤ 0.05) in double mutants in comparison to FLT3-ITD cell using single-shot LFQ and pHASED datasets. B Kinase substrate enrichment analysis (KSEA) profile of resistant cell lines compared to FLT3-ITD. Z score indicates predicted kinase activity, with a positive value predictive of kinase activation and a negative value predictive of kinase inhibition. C Reactome enrichment profile of kinases identified by KSEA as differentially regulated (log₂ ± 0.5) in resistant cell lines compared to FLT3-ITD cells. Top molecular functions and/or signaling identified by Reactome have been assigned to each cluster if significantly over-represented by kinases. D Common canonical pathways identified by Ingenuity Pathway analysis (IPA) of phosphorylated changes in resistant cell lines compared with FLT3-ITD. E Phosphorylation profile of ATM signaling substrates in resistant cell lines compared with FLT3-ITD. Yellow indicates increased phosphorylation, whereas blue represents decreased phosphorylation. F Proliferation rate after 48 h comparing FLT3-ITD/D835 to FLT3-ITD (ratio of triplicates ± SE are shown, and statistical significance was determined via ordinary one-way ANOVA, *p < 0.05). G Confirmation of differential response to treatment with tyrosine kinase inhibitor (TKI) sorafenib. Cell viability was assessed by Resazurin assay at 48 h treatment (statistical significance calculated using one-way ANOVA, *p < 0.05, **p < 0.01 and ***p < 0.001)