Fig 2: Activation of FLT3-TAZ signaling correlates with less favorable prognosis in BP-CML. A Immunoblotting analysis of FLT3-pSTAT3-TAZ components and BCR::ABL1 in 27 clinical samples derived from CP-CML patients. BP-CML patient sample 2028 was used as a control for comparing protein expression levels between CP and BP patients. CML prognostic factors such as disease phase, BCR::ABL1/ABL1 (%IS), and blast % are indicated above each patient specimen. B Immunoblotting analysis of 27 clinical samples derived from BP-CML patients. CP-CML patient sample 903 was used as a control for comparing protein expression levels between CP and BP patients. C and D Immunoblotting analysis of paired serial samples from individual patients. CHR, complete hematologic response; MyBC, myeloid blast crisis; NEL, no evidence of leukemia. E Pie chart showing the co-expression patterns between FLT3, pSTAT3, and TAZ proteins in BP-CML patients (n = 27). F and G Clinical relevance of FLT3 expression level and prognostic factors for CML, including BCR::ABL1/ABL1 ratio (IS%) (F) and blast percentage (G), in BP-CML patients. FLT3−, n = 13; FLT3.+, n = 14. All p-values were calculated using Student’s t-test and error bars are means ± SD. A p-value of less than 0.05 indicates a statistical difference. **p < 0.01

Fig 3: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrcK295N-dsRed (middle panel) or vSrcY416F-dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrcK295N-dsRed or vSrcY416F-dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY412-cABL, cABL, pY694/699-STAT5A/B, STAT5A, pY416-Src and Src. α-Tubulin served as a loading control.

Fig 4: Novel mechanisms for regulation of actin dynamics. Upon agonist activation, c-Abl mediates CAS phosphorylation, which promotes the association of CAS with Pfn-1 to activate Pfn-1. In addition, c-Abl also catalyzes cortactin phosphorylation and increases the coupling of cortactin with Pfn-1. Activated Pfn-1 facilitates actin polymerization. Src mediates c-Abl activation in response to contractile stimulation. Furthermore, actin polymerization provides a positive feedback for c-Abl activation

Fig 5: TrkA-ERK1/2-ABL1 cascade phosphorylates PHF5A.A HEK293T cells expressing Flag-PHF5A were treated with or without 60 μM ERK1/2 inhibitor magnolin for 1 h. Immunoprecipitated Flag-PHF5A was detected by a pan-tyrosine-phosphorylation antibody. B Immunoprecipitated Flag-PHF5A and whole-cell lysate co-expressed with TrkA were incubated with or without CIP (5 u) for 30 min at 37 °C. C HEK293T cells expressing Flag-PHF5A were treated with or without 5 nM ABL1 inhibitor dasatinib for 1 h. D Flag-PHF5A and HA-Abl1 were co-expressed in HEK293T cells. Immunoprecipitation of PHF5A were immunoblotted and detected by pan anti-tyrosine-phosphorylation antibody. E Bacterially purified His-PHF5A was incubated with or without HA-Abl1(wild-type, constitutively activate mutant PP, and kinase-inactive mutant K290R) purified from HEK293T cells for an in vitro kinase assay. F Y36F or Y51F was co-expressed with Abl1 in HEK293T cells. Using pY36 to detect phosphorylation of PHF5A-Y36. G Immunoprecipitated Flag-PHF5A and whole-cell lysate co-expressed with Abl1 were incubated with or without CIP (5 u) as previously. H, I HEK293T cells were transfected with indicated plasmids. Using anti-HA agarose beads (H) and anti-HA magnetic beads (I) to immunoprecipitate HA-Abl1 or HA-PHF5A. J HEK293T cells were transfected with indicated plasmids for 48 h. Immunofluorescence analyses were performed with indicated antibodies. K HEK293T cells were transfected with indicated plasmids and treated with or without MK2206 (5 μM) or magnolin (60 μM) for 1 h. Immunoblotting was performed with indicated antibodies. L Model for TrkA-ERK1/2-ABL1-PHF5A phosphorylation cascade.