Fig 1: FST was increased in FLT3/ITD‐transgenic zebrafish and FLT3/ITD‐mutated AML A–CWISH of fst in FLT3/WT (A), and FLT3/ITD plasmid DNA‐injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35).D–KGeneration and characterization of FLT3/ITD‐transgenic zebrafish. Diagrammatic representation (D and E) of the generation of Runx1‐FLT3/ITD‐transgenic zebrafish (see Materials and Methods section). GFP expression was detected by fluorescent microscopy (F–H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1‐FLT3/ITD‐transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD‐positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1‐FLT3/ITD‐transgenic zebrafish (F1) at 2 months old. Fish 4, 5, and 6 showed germline transmission of FLT3/ITD transgene (K).L–NKidney marrow (KM) was collected from Runx1‐FLT3/ITD‐transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1‐FLT3/ITD‐transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (M, N) (abbreviation for panel M: M, myeloid cells; P, progenitor cells; L, lymphoid cells; E, erythroid cells). Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum‐to‐maximum values, 25th‐to‐75th percentile, and the 50th percentile (median), respectively. **P < 0.01 (Student's t‐test).OExpression of fst was detected by RT–qPCR in KM from WT sibling and Runx1‐FLT3/ITD‐transgenic zebrafish at 18 months old. The RT–qPCR experiments were performed in triplicates, and data were presented as mean ± SEM. **P < 0.01.PDetection of FST expression, p‐ERK1/2, and p‐CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non‐specific staining of p‐ATF1 protein due to the conserved motif.Data information: Scale bar = 500 μm.Source data are available online for this figure.
Fig 2: Overexpression of FLT3/ITD induced axis duplication and ectopic expression of FST in zebrafish embryos A–DThe morphology of uninjected, FLT3/WT mRNA, and FLT3/ITD mRNA‐injected (150 ng per embryo) embryos on day 2 post‐fertilization (dpf).E–HWhole‐mount in situ hybridization (WISH) of notochord‐specific marker col9a2 in uninjected, FLT3/WT mRNA, and FLT3/ITD mRNA‐injected embryos on 2 dpf.I, JFLT3 signaling was detected by Western blotting in 293FT cells transfected with FLT3/ITD mRNA (I) or in zebrafish embryos injected with FLT3/ITD mRNA (J).KThe effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis duplication phenotype induced by FLT3/ITD mRNA injection in zebrafish.L–NQuantification of fst expression by RT–qPCR (L), Western blotting (M), and WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm. In (K and L), the experiments were performed in triplicates and the data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 (Student's t‐test). NS, not significant.Source data are available online for this figure.
Fig 3: Schematic diagram depicting the molecular mechanism of FST upregulation in FLT3/ITD‐transgenic zebrafish, knock‐in mice, and human AML Left panel. Transient overexpression of human FLT3/ITD mutation resulted in axis duplication and dorsalization abnormalities in zebrafish accompanied by upregulation of embryonic morphogen Fst. Upregulation of FST was consistently found in FLT3/ITD‐transgenic zebrafish, Flt3/ITD knock‐in mice, FLT3/ITD‐mutated AML cell lines, and primary AML samples in vitro and in vivo. Right panel. A novel FLT3/ITD‐p90RSK‐CREB‐FST signaling cascade was demonstrated in human AML. FST is a promising biomarker and therapeutic target for human FLT3/ITD+ AML.
Fig 4: FLT3/ITD upregulated FST through phosphorylation of CREB A In silico analysis (DECipherment of DNA Elements, SABiosciences) and schematic model of transcription factor binding sites on human FST promoter. CBP: CREB‐binding protein; CRE: cAMP‐response element; TSS: transcription start site.B, CThe direct binding of p‐CREB to human FST promoter was detected by ChIP‐PCR (B) and ChIP‐qPCR (C). c‐Fos was used as positive control of p‐CREB target gene. Normal IgG was used as negative control of ChIP.DDual‐luciferase assay demonstrating the direct binding of p‐CREB on human FST promoter. pRL‐CMV, Renilla luciferase vector; pGL‐CRE− and pGL‐CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE−) or wild type (CRE+); p‐GFPSpark, GFP‐expressing vector; p‐CREBY134F, CREBY134F‐GFP‐expressing vector.EFST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3‐parental (P in short) and Ba/F3‐FLT3/ITD (ITD in short) cells.F–HPhospho‐flow analysis of p‐CREB in Ba/F3‐parental, Ba/F3‐FLT3/ITD, and Ba/F3‐FLT3/ITD cells treated with FLT3 inhibitor quizartinib (Qui in short). Isotype antibody was used as control to calculate the mean fluorescence intensity (MFI) ratio (F, G). The transcription and expression of Fst were detected by RT–qPCR after quizartinib treatment (10 nM) in Ba/F3‐FLT3/ITD cells for 1 day (H).I–KThe expression of FST and phosphorylation of CREB were detected by Western blotting (I and K) and phospho‐flow analysis (J) in MOLM‐13 (I) and Ba/F3‐FLT3/ITD (K) cells treated with quizartinib and BRD7389 for 1 day, respectively.LRSK expression and FST expression were detected by Western blotting after p90RSK knockout by CRISPR/Cas9 in MOLM‐13 cells.MThe phosphorylation of CREB and FST expression was detected by Western blotting in Ba/F3‐FLT3/ITD cells treated with CREB inhibitor 666‐15 for 1 day. ^: non‐specific staining of p‐ATF1 protein due to the conserved motif.NCREB expression and FST expression were detected by Western blotting after CREB knockout by CRISPR/Cas9 in MOLM‐13 cells.OThe growth of Ba/F3‐parental (with IL‐3), Ba/F3‐FLT3/ITD (without IL‐3), and Ba/F3‐FLT3/ITD (with IL‐3) cells was measured after 3 days treatment of CREB inhibitor 666‐15 in vitro.PThe rescue effect of CREB inhibitor 666‐15 on FLT3/ITD‐induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.Data information: In (C, D, G, H, J, and O), the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P < 0.01 and ***P < 0.001 (Student's t‐test).Source data are available online for this figure.
Fig 5: FST knockdown reduced AML cell growth in vitro and in vivo A FST knockdown in MOLM‐13 by shRNA effectively reduced FST expression.B–EThe morphology (B), apoptosis (C), and clonogenicity of MOLM‐13 (D and E) were measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates.F, GThe engraftment of MOLM‐13 after FST knockdown was detected by flow cytometry of human CD45‐ and mouse CD45.1‐positive cells in recipient mouse BM aspiration at week 2 post‐transplantation.HThe effect of FST knockdown on the survival of NSG mice engrafted with MOLM‐13. scr: scrambled sequence control (7 mice); sh: short hairpin RNA (8 mice for sh1 and sh2, respectively). The survival curve was analyzed by log‐rank test. **P < 0.01.Data information: In (C, E, and G), data were presented as mean ± SEM. *P < 0.05 and **P < 0.01 (Student's t‐test).Source data are available online for this figure.
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