Journal: Cell Death & Disease

Article Title: Targeting oncogenic activation of FLT3/SREBP/FASN promotes the therapeutic effect of quizartinib involving disruption of mitochondrial phospholipids

doi: 10.1038/s41419-025-07661-6

Figure Lengend Snippet: Relative mRNA levels of major enzymes in the fatty acid synthesis pathway from FLT3-ITD cell lines ( A ) and FLT3-WT cell lines ( B ). Ctrl, control cells without treatment. Quizar_6 h, treatment with quizartinib (10 nM) for 6 h. Quizar_12 h, treatment with quizartinib (10 nM) for 12 h. Bars, means ± SD, n = 3 per group. C KEGG metabolism related pathways of downregulated genes in CD45 positive cells after quizartinib treatment in GSE202222 dataset. Arrows, lipid metabolism pathways. D Gene set enrichment analysis of the KEGG signature fatty acid metabolism (KEGG ID: hsa01212) after quizartinib treatment in GSE202222 dataset. E Volcano plot of differentially expressed genes in GSE202222. Labeled genes were those downregulated in the fatty acid or lipid metabolism after quizartinib treatment ( p < 0.05, |Fold Change| > 1.5).

Article Snippet: The treatment of quizartinib and fatostatin was as the following: (1) Vehicle control (PBS containing 10% DMSO and 10% castor-oil); (2) 15 mg/kg fatostatin by intraperitoneal injection (dissolved in PBS containing 10% DMSO and 10% castor-oil); (3) 1 mg/kg quizartinib by oral gavage (dissolved in ddH 2 O) (MedChemExpress, Monmouth Junction, NJ, USA); (4) fatostatin + quizartinib.

Techniques: Control, Labeling

Journal: Cell Death & Disease

Article Title: Targeting oncogenic activation of FLT3/SREBP/FASN promotes the therapeutic effect of quizartinib involving disruption of mitochondrial phospholipids

doi: 10.1038/s41419-025-07661-6

Figure Lengend Snippet: Relative Srebf1 / SREBF1 mRNA level in murine ( A ) and human ( B ) leukemia cell lines with control, quizartinib (10 nM) treatment for 6 h and 12 h. N = 3 replicates. Bars, means ± S.D. C Upper panel, western blotting of SREBP1 and FASN in FLT3-WT and FLT3-ITD cell lines before and after quizartinib treatment of 6 and 12 h. P precursor form, N nuclear form. Lower panel, comparison of SREBP1 protein expression in the FLT3/ITD cells before and after quizartinib treatment. N = 3 replicates. Bars, means ± S.D. D Log2CPM gene-expression values of SREBF1 / Srebf1 in TCGA_LAML, GSE163926 and GSE202222 datasets. E The polysome profile of BaF3/ITD cells. 40S, 60S, and 80S ribosomal subunits and polysomes were fractionated using 5–50% density gradient sucrose buffer and monitored with A260 measurements. F , G Total RNA of BaF3/ITD cells before and after quizartinib treatment was extracted from each fraction. The expression of Srebf1 and Actb was measured by qPCR. The mRNA content from monosomes (40S, 60S, 80S) represent the non-translated portion. The mRNA content from polysome represents the translated portion. N = 3 independent experiments. Bars, means ± S.D.

Article Snippet: The treatment of quizartinib and fatostatin was as the following: (1) Vehicle control (PBS containing 10% DMSO and 10% castor-oil); (2) 15 mg/kg fatostatin by intraperitoneal injection (dissolved in PBS containing 10% DMSO and 10% castor-oil); (3) 1 mg/kg quizartinib by oral gavage (dissolved in ddH 2 O) (MedChemExpress, Monmouth Junction, NJ, USA); (4) fatostatin + quizartinib.

Techniques: Control, Western Blot, Comparison, Expressing, Gene Expression

Journal: Cell Death & Disease

Article Title: Targeting oncogenic activation of FLT3/SREBP/FASN promotes the therapeutic effect of quizartinib involving disruption of mitochondrial phospholipids

doi: 10.1038/s41419-025-07661-6

Figure Lengend Snippet: A BaF3/ITD were pre-treated with vehicle control (DMSO) or quizartinib (10 nM) for 6 h. SREBP1 (precursor) protein expression was then detected after treatment with cycloheximide (CHX, 100 μg/mL) at the indicated time points. B The plotted graph of three independent experiments showing the relative amount of SREBP1 at each time point compared to the control without CHX treatment. C Western blotting showing SREBP1 protein expression in the presence of CHX (100 μg/mL) and MG132 (10 μM). D Expression of total AKT, phosphor-AKT (p-AKT S473 ), SREBP1 and FASN in BaF3/ITD and MV4-11 cells before and after treatment of MK2206 (5 μM) for 6 and 12 h. E Expression of total AKT, phospho-AKT (p-AKT S473 ), total GSK3β and phospho-GSK3β (p-GSK3β S9 ) in BaF3/ITD and MV4-11 cells before and after treatment of quizartinib (10 nM) for 6 and 12 h. Vinculin was used as loading control. F Expression of SREBP1 and FASN in BaF3/ITD and MV4-11 cells before and after treatment of CHIR-99021 (3 μM) for 6 and 12 h. G The ubiquitination and phosphorylation (pan phospho-Ser/Thr) levels of SREBP1 before and after treatment of CHIR-99021 for 6 h. IP immunoprecipitation. Total input of SREBP1 and α-Tubulin was shown as equal loading. H Expression of SREBP1in BaF3/ITD and MV4-11 cells with shRNA targeting GSK3β . NC non-specific control. Sh #1 and Sh #2, different sequencing of shRNA targeting GSK3β . I The ubiquitination and phosphorylation (pan phospho-Ser/Thr) levels of SREBP1 before and after treatment of quizartinib for 6 h. J An illustrated pathway displaying the regulation of protein degradation of SREBP by the FLT3/AKT/GSKβ phosphorylation cascade and the kinase inhibitors including quizartinib, MK2206 and CHIR-99021.

Article Snippet: The treatment of quizartinib and fatostatin was as the following: (1) Vehicle control (PBS containing 10% DMSO and 10% castor-oil); (2) 15 mg/kg fatostatin by intraperitoneal injection (dissolved in PBS containing 10% DMSO and 10% castor-oil); (3) 1 mg/kg quizartinib by oral gavage (dissolved in ddH 2 O) (MedChemExpress, Monmouth Junction, NJ, USA); (4) fatostatin + quizartinib.

Techniques: Control, Expressing, Western Blot, Immunoprecipitation, shRNA, Sequencing

Journal: Cell Death & Disease

Article Title: Targeting oncogenic activation of FLT3/SREBP/FASN promotes the therapeutic effect of quizartinib involving disruption of mitochondrial phospholipids

doi: 10.1038/s41419-025-07661-6

Figure Lengend Snippet: A , B The FLT3-ITD leukemia cell lines were treated with vehicle or quizartinib (10 nM) at the indicated time points. Mitochondrial membrane potential and cell death was assessed by Rho123 and Annexin V/PI, respectively. n = 3 per group. Bars, means ± S.D. C Lipidomic analysis of MOLM-13 cells before and after treatment of quizartinib (10 nM) for 12 h. Significant differentially abundant metabolites between two groups ( p < 0.05, |Log2FC | > 1 and VIP > 1) are shown in red (upregulated) and blue (downregulated) in the volcano plot. D The pie graph illustrating the proportion of each lipid class contributing to the total number of upregulated (red) and downregulated (blue) features. E The illustration of enzymatic steps involved in biosynthesis of major phospholipids. F Comparison of lipid abundance between the control and treatment group in all lipid classes with statistical significance. Blue stars, major types of phospholipids involved in the biosynthesis pathway. N = 6 replicates. Bars, means ± S.D. ***( p < 0.001) and ****( p < 0.0001) indicate p values for the major phospholipids with blue stars. p < 0.05 for all other lipid species. G The absolute cardiolipin contents in FLT3/ITD cells treated before and after quizartinib (10 nM, 12 h) were detected by the fluorometric assay as described in the method. N = 3 replicates. Bars, means ± S.D. The abbreviations of all lipid species and enzymes were listed in the Supplementary Table .

Article Snippet: The treatment of quizartinib and fatostatin was as the following: (1) Vehicle control (PBS containing 10% DMSO and 10% castor-oil); (2) 15 mg/kg fatostatin by intraperitoneal injection (dissolved in PBS containing 10% DMSO and 10% castor-oil); (3) 1 mg/kg quizartinib by oral gavage (dissolved in ddH 2 O) (MedChemExpress, Monmouth Junction, NJ, USA); (4) fatostatin + quizartinib.

Techniques: Membrane, Comparison, Control

Journal: Cell Death & Disease

Article Title: Targeting oncogenic activation of FLT3/SREBP/FASN promotes the therapeutic effect of quizartinib involving disruption of mitochondrial phospholipids

doi: 10.1038/s41419-025-07661-6

Figure Lengend Snippet: A , B Quantitative analysis of cell death after different drug treatment for 48 h in FLT3/ITD (BaF3/ITD, MOLM-13) and FLT3-WT (BaF3, HL-60) cells. N = 3 replicates. Bars, means ± S.D. Ctrl control cells treated with vehicle (DMSO), Orlis orlistat (10 μM), Quizar quizartinib (10 nM), Fato fatostatin (5 μM). C , D Western blot analysis showing knockdown of SREBP1 and FASN protein in BaF3/ITD and MOLM-13 cells by shRNA. NC non-specific shRNA sequence. E , F Quantitative analysis of cell death in BaF3/ITD and MOLM-13 cells following quizartinib treatment, with or without SREBP and FASN knockdown by shRNA.

Article Snippet: The treatment of quizartinib and fatostatin was as the following: (1) Vehicle control (PBS containing 10% DMSO and 10% castor-oil); (2) 15 mg/kg fatostatin by intraperitoneal injection (dissolved in PBS containing 10% DMSO and 10% castor-oil); (3) 1 mg/kg quizartinib by oral gavage (dissolved in ddH 2 O) (MedChemExpress, Monmouth Junction, NJ, USA); (4) fatostatin + quizartinib.

Techniques: Control, Western Blot, Knockdown, shRNA, Sequencing

Journal: Cell Death & Disease

Article Title: Targeting oncogenic activation of FLT3/SREBP/FASN promotes the therapeutic effect of quizartinib involving disruption of mitochondrial phospholipids

doi: 10.1038/s41419-025-07661-6

Figure Lengend Snippet: A Illustration of the animal experiment: 2 days after tail vein injection of BaF3/ITD-EGFP-LUC2 cells, BALB/c mice were randomized and treated with fatostatin (15 mg/kg i.p. daily), orlistat (240 mg/kg i.p. daily), quizartinib (1 mg/kg oral daily), or quizartinib plus fatostatin/orlistat for another 28 days. B , C Leukemia burden and progression was monitored by luminescence imaging every 4 days at early stage and every week at late stage. n = 3 mice per group. D , E Survival was estimated by Kaplan–Meier analysis as described in Methods. n = 11 mice in each group. F , G Measurement of body weights of each group. Blank normal mice without leukemia cell injection, Ctrl control group with leukemia burden, Orlis orlistat, Fato fatostatin, Quizar quizartinib.

Article Snippet: The treatment of quizartinib and fatostatin was as the following: (1) Vehicle control (PBS containing 10% DMSO and 10% castor-oil); (2) 15 mg/kg fatostatin by intraperitoneal injection (dissolved in PBS containing 10% DMSO and 10% castor-oil); (3) 1 mg/kg quizartinib by oral gavage (dissolved in ddH 2 O) (MedChemExpress, Monmouth Junction, NJ, USA); (4) fatostatin + quizartinib.

Techniques: Injection, Imaging, Control

Journal: Marine Drugs

Article Title: The Novel Diketopiperazine Derivative, Compound 5-3, Selectively Inhibited the Proliferation of FLT3-ITD Mutant Acute Myeloid Leukemia (AML) Cells

doi: 10.3390/md23070289

Figure Lengend Snippet: The impact of compound 5-3 on the FLT3 pathway in FLT3-ITD mutant cells. ( A , B ) The FLT3-ITD overexpressing BaF3 cells were treated with a range of concentrations (6.25, 12.5, 25, 50, and 100 nM) of compound 5-3 for 4 h. Subsequently, the whole-cell lysates were prepared and subjected to Western blot analysis to detect the phosphorylation levels of FLT3 and its downstream signaling molecules. ( C , D ) The FLT3-ITD mutant MV4-11 cells were treated with a series of concentrations (20, 100 and 500 nM) of compound 5-3 for 4 h. Subsequently, the proteins were extracted for Western blot analysis. ( E , F ) The FLT3-ITD mutant MV4-11 cells were treated with compound 5-3 (1000 nM) or AC220 (100 nM) for 12 and 24 h, respectively, and then the cDNA were extracted for RT-PCR analysis.

Article Snippet: AC220 were purchased from Aladdin Scientific (ShangHai, China).

Techniques: Mutagenesis, Western Blot, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction

Journal: Marine Drugs

Article Title: The Novel Diketopiperazine Derivative, Compound 5-3, Selectively Inhibited the Proliferation of FLT3-ITD Mutant Acute Myeloid Leukemia (AML) Cells

doi: 10.3390/md23070289

Figure Lengend Snippet: Effects of 5-3 on the phosphorylation of c-KIT. ( A ) The c-KIT overexpressing Kasumi-1 cells were treated with different concentrations of compound 5-3 and AC220 for 48 h, respectively. After treatment, cell viability was assessed by the MTT assay. ( B ) Kasumi-1 cells were treated with 5-3 and AC220 at different concentrations for 4 h, and then whole-cell lysates were subjected to Western blot analysis to detect the phosphorylation of c-KIT.

Article Snippet: AC220 were purchased from Aladdin Scientific (ShangHai, China).

Techniques: Phospho-proteomics, MTT Assay, Western Blot

Journal: Marine Drugs

Article Title: The Novel Diketopiperazine Derivative, Compound 5-3, Selectively Inhibited the Proliferation of FLT3-ITD Mutant Acute Myeloid Leukemia (AML) Cells

doi: 10.3390/md23070289

Figure Lengend Snippet: Binding mode of compound 5-3 within the crystal structure of FLT3 (PDB: 4XUF). ( A ) Compound 5-3 (orange sticks) and AC220 (blue sticks) are depicted along with the surface representation of the binding site. ( B ) The detailed view of the interactions is presented; side chains of residues involved in hydrophobic or π-interactions are depicted as gray sticks. Amino acid residues forming hydrogen bonds are illustrated with both main and side chains represented as cyan sticks. Other atoms are colored as follows: nitrogens (blue), oxygens (red), sulfur (yellow) and polar hydrogens (white).

Article Snippet: AC220 were purchased from Aladdin Scientific (ShangHai, China).

Techniques: Binding Assay

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) MOLM14 cells were added to a 384-well plate containing cytokines, chemokines, and growth factors with 10 nM AC220 (quizartinib). Viability was measured using MTS reagent, averaged over all concentrations (1, 10, and 100 ng/ml), and the results sorted according to highest average viability. Average viability >2 standard deviations above the mean are highlighted in red and by an *. B) MOLM14 cells were cultured in lower concentrations of recombinant proteins (1, and 0.1 ng/ml) in 96-well plates plus 10 nM AC220. Viability was assessed after 48 hours with MTS reagent and data plotted as percent of the respective untreated control. All wells were plated in triplicate with standard deviation indicated by error bars. C) MOLM14 cells were cultured continuously in 10 nM AC220 with FL, FGF2, or media alone as indicated (N=4 for each). Fresh media, AC220 and cytokine were replaced every 2–3 days over the indicated time period. Viable cell numbers were analyzed every 2–3 days with Guava ViaCount.

Article Snippet: Tyrosine kinase inhibitors Quizartinib (AC220) and sorafenib were purchased from LC labs (Woburn, MA, USA).

Techniques: Cell Culture, Recombinant, Control, Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: The time to development of exponential growth (indicated by gray) was plotted over time for the extended cultures in Figure 1C. The timepoints at which Sanger or next-generation sequencing (at right, average read depth ~1500) was performed is indicated by arrows. If a mutation could not be detected it is indicated with “−”. A) MOLM14 cells cultured in media and AC220 alone (no ligand) developed exponential growth later than FGF2- and FL-dependent cultures (panels B and C) and developed early resistance mutations: D839V and D835H. B) FGF2-dependent and C) FL-dependent resistant cultures (cultured continuously in 10 ng/ml FGF2 or FL plus 10 nM AC220) were split at 4 months and 1×107 cells placed into fresh media without recombinant protein with continued 10 nM AC220 treatment. Fresh media and 10 nM AC220 were replaced every 2–3 days over the indicated time period and viable cell number was analyzed using Guava ViaCount. D) Naïve MOLM14 cells and resistant MOLM14 cells with FLT3 D839V mutation (R-1), FLT3 D842C mutation (FGF2 R-1), and non-mutated resistant cells (FL R1 after FL subtraction) were exposed to a gradient of FLT3 inhibitors: AC220 (quizartinib), crenolanib, sorafenib, and ponatinib. Viability was measured after 48 hours by MTS assay with average viability scaled relative to untreated cells (100%). The experiment was done in triplicate and error bars represent standard deviation.

Article Snippet: Tyrosine kinase inhibitors Quizartinib (AC220) and sorafenib were purchased from LC labs (Woburn, MA, USA).

Techniques: Next-Generation Sequencing, Mutagenesis, Cell Culture, Recombinant, MTS Assay, Standard Deviation

Journal: Theranostics

Article Title: LT-171-861, a novel FLT3 inhibitor, shows excellent preclinical efficacy for the treatment of FLT3 mutant acute myeloid leukemia

doi: 10.7150/thno.46593

Figure Lengend Snippet: Potency comparison of FLT3 inhibitors in cellular assays

Article Snippet: Sorafenib (Cat. #3009) and AC220 (Cat. #5793) were purchased from ApexBio Company (Houston, TX).

Techniques: Comparison

Journal: Theranostics

Article Title: LT-171-861, a novel FLT3 inhibitor, shows excellent preclinical efficacy for the treatment of FLT3 mutant acute myeloid leukemia

doi: 10.7150/thno.46593

Figure Lengend Snippet: Potent pro-apoptotic ability of LT-171-861 in leukemia cells harboring FLT3-ITD mutation. ( A ) MOLM13 cells and MV4-11 cells were respectively incubated with indicated concentrations of LT-171-861, sorafenib and AC220 for 2 h. Cell lysates were used to perform western blot assay. ( B ) MV4-11 cells and MOLM13 cells were treated with LT-171-861 for 36 h. Cells then stained with propidium iodide (PI) /annexin V-FITC and then evaluated by flow cytometry. Annexin V + /PI - and annexin V + /PI + cells were considered dead cells and shown in the bottom chart (n=3). ( C ) MOLM13 cells and MV4-11 cells were treated with indicated concentrations of LT-171-861 for 36 h. PARP, caspase 3, caspase 8, Mcl-1 were evaluated in the cell lysate by western blot. ( D ) MV4-11 cells were treated with LT-171-861 (200 nM), AC220 (200nM) or sorafenib (200 nM) for indicated time periods (0, 4, 8, 12, 24, and 36 h) respectively. Then Cells were stained with propidium iodide/annexin V-FITC and evaluated by flow cytometry. ( E ) MOLM13 cells and MV4-11 cells were treated with cytarabine, LT-171-861 or a combination of cytarabine and LT-171-861 (ratio of 10:1), and cell viability was measured by MTT assay. Error bars show standard deviation (SD), and the combination index (CI) value was calculated by CompuSyn 1.0 software.

Article Snippet: Sorafenib (Cat. #3009) and AC220 (Cat. #5793) were purchased from ApexBio Company (Houston, TX).

Techniques: Mutagenesis, Incubation, Western Blot, Staining, Flow Cytometry, MTT Assay, Standard Deviation, Software

Journal: Theranostics

Article Title: LT-171-861, a novel FLT3 inhibitor, shows excellent preclinical efficacy for the treatment of FLT3 mutant acute myeloid leukemia

doi: 10.7150/thno.46593

Figure Lengend Snippet: Inhibition of FLT3 in BaF3 cell models and in stroma-protecting setting. ( A ) Indicated FLT3 mutations were expressed in BaF3 cells respectively. Transformed BaF3 cells were incubated for 2 h with indicated concentrations of LT-171-861, and ( B-E ) for direct comparison with 3 inhibitors. The autophosphorylation level of FLT3 was visualized and compared by western blot analysis. (E) MOLM13 cells were treated with indicated concentrations of LT-171-861, sorafenib and AC220 for 72 h in condition medium (CM) and normal culture medium (NM). The CM was prepared from HS-5 cell culture for 4 days under routine culture conditions, clarified by centrifugation, and used immediately. The CM was added to complete medium at a final concentration of 40%. Cell viabilities were measured by MTT assay. Data were shown as mean±SD. ( F ) MOLM13 cells were incubated for 8 h in CM with indicated concentrations of LT-171-861 and AC220. The FLT3 phosphorylation levels of FLT3, STAT5 and ERK 1/2 were determined by Western blot.

Article Snippet: Sorafenib (Cat. #3009) and AC220 (Cat. #5793) were purchased from ApexBio Company (Houston, TX).

Techniques: Inhibition, Transformation Assay, Incubation, Comparison, Western Blot, Cell Culture, Centrifugation, Concentration Assay, MTT Assay, Phospho-proteomics

Journal: Theranostics

Article Title: LT-171-861, a novel FLT3 inhibitor, shows excellent preclinical efficacy for the treatment of FLT3 mutant acute myeloid leukemia

doi: 10.7150/thno.46593

Figure Lengend Snippet: PK/PD and preliminary toxicity evaluations of LT-171-861. ( A ) Concentrations of LT-171-861 in plasma collected at indicated time points were measured by HPLC/LC-MAS analysis. Blue square and solid line represent intravenous injection administration. ( B ) PD study of LT-171-861 in BALB/c nude mice bearing tumors (120 mm 3 ). Mice were sacrificed at indicated time points and then tumor tissues were dissected and lysed. Phosphorylation levels of FLT3, STAT5 and ERK1/2 were measured by western blotting. ( C ) Bone marrow mononuclear cells (BMMCs) were isolated from healthy C57BL/6 mice. Cells were treated with indicated concentrations of LT-171-861. Apoptosis assays were conducted after 36-hour incubation. ( D ) Healthy C57BL/6 mice (n=6) were intravenously injected with vehicle or LT-171-861 (10 mg/kg) every other day and were orally administered with AC220 (20 mg/kg) or sorafenib (20 mg/kg) once per day for 18 days. 24 h after the last administration, c-Kit + cells were evaluated by flow cytometry. ( E ) PBMCs collected from 3 healthy donors, were incubated with LT-171-861 at indicated concentrations for 72 h and cell viabilities were conducted to evaluate by MTT assay. Data were shown with mean±SD.

Article Snippet: Sorafenib (Cat. #3009) and AC220 (Cat. #5793) were purchased from ApexBio Company (Houston, TX).

Techniques: Clinical Proteomics, Injection, Phospho-proteomics, Western Blot, Isolation, Incubation, Flow Cytometry, MTT Assay

Journal: Theranostics

Article Title: LT-171-861, a novel FLT3 inhibitor, shows excellent preclinical efficacy for the treatment of FLT3 mutant acute myeloid leukemia

doi: 10.7150/thno.46593

Figure Lengend Snippet: Inhibitory efficacy of LT-171-861 against patient blasts. ( A ) AML blasts harboring FLT3-ITD (patient 1 and 5) and FLT3-wt (patient 2-4 and 6) were treated with indicated concentrations of LT-171-861 for 36 h. Cells then stained with propidium iodide (PI)/annexin V and then evaluated by flow cytometry. ( B ) AML patient blasts expressing FLT3-ITD (patient 7, 9 and 10) were incubated with indicated concentrations of LT-171-861, sorafenib and AC220 for 72 h and cell viabilities were evaluated with MTT assay. ( C ) Blasts from AML patients harboring FLT3-ITD (patient 7 and 8) were incubated with LT-171-861 for 2 h, and p-FLT3, p-STAT5 and p-ERK1/2 were tested by western blot.

Article Snippet: Sorafenib (Cat. #3009) and AC220 (Cat. #5793) were purchased from ApexBio Company (Houston, TX).

Techniques: Staining, Flow Cytometry, Expressing, Incubation, MTT Assay, Western Blot