Use of sgi-7079 in the treatment of flt3-mutant acute myeloid leukemia

Abstract

The application discloses application of SGI-7079 in treating FLT3 mutant acute myeloid leukemia. The application research shows that SGI-7079 can be stably combined with FLT3, inhibit the phosphorylation level of FLT3 and downstream STAT5, AKT and ERK, and induce G1 phase arrest and cell apoptosis. In vitro, SGI-7079 has strong inhibitory effect on FLT3-ITD and drug-resistant mutant cells; in a FLT3-ITD mouse model, SGI-7079 can significantly reduce leukemia load and prolong survival, and shows better curative effect than existing drugs on drug-resistant mutations such as F691L and D835Y. Meanwhile, SGI-7079 also has significant inhibitory effect on primary cells of FLT3-ITD positive patients. It is shown that SGI-7079 can be used as a new drug candidate for treating FLT3 mutation and drug-resistant AML, and provides a new direction for optimizing targeted therapy strategy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of SGI-7079 in the treatment of FLT3-mutant acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant hematologic disorder characterized by uncontrolled proliferation and arrested differentiation of myeloid progenitor cells, exhibiting significant heterogeneity and aggressive clinical behavior. Although targeted therapy and hematopoietic stem cell transplantation have improved the prognosis of some patients to some extent, the overall cure rate remains low, and drug resistance-induced relapse continues to pose a significant clinical challenge. FLT3 (Fms-like tyrosine kinase 3) is a receptor tyrosine kinase that is mutated in approximately one-third of AML cases, making it one of the most common genetic disorders. FLT3 mutations are closely associated with disease progression, relapse, and poor prognosis. FLT3 mutations mainly include internal tandem repeat mutations (ITD) (FLT3-ITD) and tyrosine kinase domain mutations (TKD) (FLT3-TKD). ITD mutations mainly occur in the proximal membrane region, disrupting the enzyme's self-inhibitory conformation, inducing ligand-independent receptor dimerization, and persistently activating the STAT5, PI3K / AKT, and MAPK / ERK pathways, thereby promoting leukemia cell proliferation, accelerating disease progression, and leading to poor prognosis. TKD mutations mainly involve amino acid substitutions of key residues D835 or gate residue F691 within the kinase domain. They often occur as a secondary resistance mechanism after FLT3 inhibitor treatment, and their impact on prognosis remains controversial in different studies.

[0003] FLT3 tyrosine kinase inhibitors (TKIs) have become an important targeted therapy for AML. First-generation FLT3 inhibitors are multi-target tyrosine kinase inhibitors, including tandutinib, lettatinib, sunitinib, midostaurin, and sorafenib. Combined with chemotherapy, they can improve the prognosis of newly diagnosed patients, but their low selectivity leads to dose limitations, significant toxic side effects, and difficulty in effectively overcoming resistance mutations. Second-generation FLT3 inhibitors, such as gilteritinib, quizartinib, and crenolanib, have significantly improved targeting selectivity, effectively addressing specific resistance mutations and showing enhanced efficacy in relapsed or refractory AML patients. Gilteritinib has been approved for monotherapy in patients with relapsed or refractory FLT3-mutant AML, significantly prolonging overall survival. FLT3 inhibitors are classified into type I and type II based on their binding characteristics to the kinase conformation. Type I inhibitors bind to the active kinase conformation, effectively inhibiting ITD and TKD mutations, while type II inhibitors act on the inactive conformation, primarily targeting ITD mutations with limited inhibitory effect on TKD mutations. Although second-generation FLT3 inhibitors have shown improved efficacy, secondary resistance mutations, especially the gate mutation F691L and the kinase domain mutation D835, remain the main mechanisms leading to the ineffectiveness of many FLT3 inhibitors. Furthermore, the toxic side effects and pharmacokinetic limitations of some inhibitors also restrict their long-term clinical application. Therefore, developing novel FLT3 inhibitors capable of overcoming resistance mutations is of great significance for improving the treatment outcomes and clinical prognosis of AML patients.

[0004] SGI-7079 is a selective, ATP-competitive, orally effective receptor tyrosine kinase Axl inhibitor that blocks Axl-mediated NF-κB activation and MMP-9 expression, thereby inhibiting tumor cell proliferation, migration, and invasion. It has demonstrated significant antitumor activity in various solid tumor models. However, the therapeutic effect of SGI-7079 in AML, especially FLT3-mutant AML, has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of SGI-7079 in the preparation of a therapeutic drug for treating FLT3-mutant acute myeloid leukemia.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: FLT3-ITD mutation is one of the common gene abnormalities in AML, often leading to poor prognosis and relapse. The efficacy of existing FLT3 inhibitors is easily limited by secondary TKD mutations. This invention, through extensive in vitro and in vivo experiments, found that SGI-7079 can significantly inhibit the proliferation of FLT3-mutant positive leukemia cells, block downstream signaling pathways, induce apoptosis, and prolong the survival of mice carrying FLT3 mutations, demonstrating its potential application value in the development of drugs for the treatment of FLT3-driven leukemia.

[0007] Specifically, this study shows that SGI-7079 can stably bind to FLT3, inhibiting the phosphorylation levels of FLT3 and its downstream STAT5, AKT, and ERK, and inducing G1 phase arrest and apoptosis. In vitro, SGI-7079 exhibits potent inhibitory effects on FLT3-ITD and its secondary TKD resistance mutations. In the FLT3-ITD mouse model, it significantly reduces leukemia burden and prolongs survival, and shows superior efficacy against secondary TKD resistance mutations such as F691L, D835Y, D835V, D835F, and Y842C compared to currently approved clinical FLT3 inhibitors. Simultaneously, SGI-7079 also shows significant inhibitory effects in primary AML cells from FLT3-ITD-positive patients, exhibiting superior inhibitory effects compared to existing clinical FLT3 inhibitors. These findings suggest that SGI-7079 could be a potential new drug candidate for treating FLT3 mutations and their secondary TKD resistance, and provide a new direction for optimizing targeted therapy strategies.

[0008] Therefore, the present invention provides the use of SGI-7079 in the preparation of a medicament for treating FLT3-mutant acute myeloid leukemia, wherein the structural formula of SGI-7079 is shown in formula (I): The FLT3 mutation is an FLT3-ITD mutation, an FLT3-TKD mutation, or an FLT3-ITD mutation accompanied by TKD resistance. That is, it includes FLT3-ITD mutation only, FLT3-TKD mutation only, or secondary resistance mutations of FLT3-ITD+TKD.

[0009] Furthermore, the FLT3-ITD-associated TKD resistance mutation is selected from one or more of the following resistance mutations: D835Y, D835V, D835F, Y842C, or F691L.

[0010] Preferably, the FLT3-ITD-associated TKD resistance mutation is selected from D835Y and / or F691L.

[0011] Furthermore, the resistance mutation is a resistance mutation against FLT3-TKIs. FLT3-TKIs include quezartinib, midotulin, sorafenib, gipritinib, etc.

[0012] Specifically, the drug exerts its therapeutic effect by inhibiting the phosphorylation of STAT5, AKT, and ERK in FLT3 and its downstream signaling pathways.

[0013] Specifically, the drug exerts its therapeutic effect by inhibiting the proliferation of FLT3-mutant positive leukemia cells, blocking the cell cycle, and inducing apoptosis. SGI-7079 can arrest AML cells in the G1 phase, significantly increase the proportion of apoptotic cells, and effectively inhibit the activation of downstream signaling pathways such as p-FLT3, p-STAT5, p-AKT, and p-ERK, thereby blocking FLT3-mediated oncogenic signals.

[0014] Specifically, the drug exerts its therapeutic effect by inhibiting the infiltration of leukemia cells in the patient's peripheral blood, bone marrow, and spleen.

[0015] Specifically, the drug achieves high-affinity and stable binding by directly targeting and binding to the FLT3-ITD protein, thereby overcoming drug tolerance caused by FLT3-TKD resistance mutations.

[0016] Furthermore, the concentration of SGI-7079 in the drug is 1 nM to 1 μM.

[0017] Furthermore, the drug also contains a pharmaceutically acceptable carrier or excipient.

[0018] Furthermore, the dosage form of the drug is oral or injectable.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of SGI-7079 in the treatment of FLT3-mutant acute myeloid leukemia. Studies in this invention show that SGI-7079 can exert broad-spectrum and potent anti-leukemic activity against various FLT3 mutation backgrounds. Its mechanism of action includes stable binding to FLT3 kinase, inhibition of phosphorylation and downstream signaling pathways, and induction of cell cycle arrest and apoptosis. In vivo, SGI-7079 can significantly reduce leukemia burden, improve histopathological manifestations, and prolong survival, especially overcoming drug tolerance caused by drug resistance mutations (such as F691L and D835Y). Furthermore, SGI-7079 also shows significant inhibitory effects in primary AML cells from FLT3-ITD-positive patients. Moreover, SGI-7079's efficacy against FLT3-ITD and drug resistance mutations is significantly superior to that of existing drugs. This indicates that SGI-7079 not only has the potential to treat FLT3 mutations and overcome resistance to existing FLT3-TKIs, but also provides a new drug strategy for the treatment of drug-resistant AML, demonstrating its significant pharmaceutical value and clinical application prospects as a novel anti-leukemia drug. Attached Figure Description

[0020] Figure 1 This study investigated the inhibitory effect of SGI-7079 on FLT3-ITD acute myeloid leukemia (AML) cells and its induction of apoptosis. Figure 1 Figure A shows the dose-response curves and corresponding IC50 values ​​of MOLM-13 and MV4-11 cells after 48 hours of treatment with SGI-7079. 50 Value; B represents the dose-response curve and IC50 value of Ba / F3 cells with or without FLT3-ITD mutation after 48 hours of treatment with SGI-7079. 50 Values; C represents the GO and KEGG pathway enrichment analysis results of differentially expressed genes in MOLM-13 and MV4-11 cells after treatment with SGI-7079; D-E represent the cell cycle distribution of MOLM-13 and MV4-11 cells after 24 hours of treatment with 3 nM SGI-7079; F-H represent the apoptosis rate of MOLM-13 and MV4-11 cells after 48 hours of treatment with different concentrations of SGI-7079; I-J represent the protein expression of Caspase-8 cleavage products and PARP1 cleavage products in MOLM-13 and MV4-11 cells after 48 hours of treatment with different concentrations of SGI-7079.

[0021] Figure 2 This describes the binding mode of SGI-7079 to FLT3 kinase and its inhibitory effect on the FLT3 signaling pathway. Among other things, Figure 2 A represents the chemical structure of SGI-7079; B shows the predicted molecular docking of SGI-7079 at the ATP-binding pocket of the FLT3 kinase domain, illustrating key hydrogen bond interactions; C–D show the CETSA results of Ba / F3-FLT3-ITD cells treated with SGI-7079 or DMSO for 1 hour at a gradient temperature of 37°C to 55°C, including the immunoblot image (C) and melting curve (D) of FLT3 protein; E–G show the expression of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK, and ERK proteins in FLT3-ITD AML cells treated with SGI-7079 for 6 hours; H–I show the KEGG pathway enrichment analysis results of downregulated genes in MOLM-13 and MV4-11 cells treated with SGI-7079 for 24 hours.

[0022] Figure 3 The results show the anti-leukemic effect of SGI-7079 in the Ba / F3-FLT3-ITD AML mouse model. Figure 3A is a schematic diagram of the FLT3-ITD AML mouse model establishment and drug treatment process; B-D are the proportions of GFP-positive leukemia cells in the peripheral blood (B), bone marrow (C), and spleen (D) of each group of mice; E is the spleen weight of each group of mice; F is an actual image of the spleen of each group of mice; G is a representative image of H&E staining of liver and spleen tissue sections of each group of mice.

[0023] Figure 4 This study investigated the inhibitory effect of SGI-7079 on FLT3-ITD-TKD mutant cells. Figure 4 A shows the dose-response curves of Ba / F3 cells carrying different FLT3-ITD secondary resistance mutations after 48 hours of SGI-7079 treatment; B shows the IC50 values ​​of each mutant cell line after treatment with SGI-7079, gipretinib, and quezatinib. 50 Value comparison; C~G are the dose-response curves of Ba / F3 cells with different FLT3 mutations after treatment with SGI-7079, giretinib and quezatinib respectively; H~L are the expression of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK and ERK proteins in the above mutant cells after treatment with different concentrations of SGI-7079 for 6 hours.

[0024] Figure 5 The effects of SGI-7079 in the FLT3-ITD-F691L AML mouse model are shown. Figure 5 A is a schematic diagram of the model establishment and drug treatment process; B-D are the proportions of GFP-positive leukemia cells in the peripheral blood (B), bone marrow (C), and spleen (D) of each group of mice; E is the spleen weight of each group of mice; F is a representative physical image of the spleen of each group of mice; G is the survival curve of each group of mice; H is a representative image of H&E staining of liver and spleen tissue sections of each group of mice.

[0025] Figure 6 The effects of SGI-7079 in the FLT3-ITD-D835Y AML mouse model are presented. Figure 6 A is a schematic diagram of the model establishment and drug treatment process; B-D are the proportions of GFP-positive leukemia cells in the peripheral blood (B), bone marrow (C), and spleen (D) of each group of mice; E is the spleen weight of each group of mice; F is a representative physical image of the spleen of each group of mice; G is the survival curve of each group of mice; H is a representative image of H&E staining of liver and spleen tissue sections of each group of mice.

[0026] Figure 7 The study investigated the efficacy of SGI-7079 on primary cells derived from patients with FLT3-ITD acute myeloid leukemia. Among these, Figure 7In the middle A to G, the proliferation inhibition results of primary bone marrow cells from 6 patients with FLT3-ITD AML were obtained after treatment with different concentrations of SGI-7079, giretinib, or quezatinib for 48 hours. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] 1. General Materials and Commonly Used Methods 2. (1) Patient samples and primary cell cultures Bone marrow (BM) samples were collected from 6 AML patients in this study. All patients signed written informed consent forms. The study followed the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Technology. Primary leukemia cells were isolated by Lymphoprep density gradient centrifugation and cultured in RPMI-1640 medium containing 20% ​​fetal bovine serum (FBS) at 37°C under 5% CO2 humidification.

[0030] (2) Cell lines and culture conditions AML cell lines MOLM-13, MV4-11, RS411, Kasumi-1, OCI-AML3, THP-1, Reh, HL-60, and K562 were routinely cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gemini) and 1% penicillin-streptomycin at 37°C and 5% CO2 humidification. Ba / F3 and its derivative cell lines carrying FLT3 mutations (FLT3-ITD, FLT3-ITD-F691L, FLT3-ITD-D835Y, FLT3-ITD-D835V, FLT3-ITD-Y842C) were established via retroviral infection.

[0031] (3) Drugs and stock solutions SGI-7079 (CAS No: 1239875-86-5), gilteritinib, and quizartinib were purchased from TargetMol (Boston, USA). In in vitro experiments, each compound was prepared as a 10 mM DMSO stock solution and stored at -20°C. Before use, it was diluted to the working concentration, with a final DMSO concentration ≤0.1%. For in vivo administration, gilteritinib was dissolved in 5% DMSO, 35% PEG300, 10% Tween 80, and 50% sterile water; quizartinib was dissolved in 5% DMSO and 0.5% methylcellulose solution; and SGI-7079 was dissolved in 5% DMSO, 40% PEG300, and 55% (w / v) 30% cyclodextrin solution.

[0032] (4) Instruments and experimental conditions Cell culture was performed in a humidified incubator at 37°C and 5% CO2. Cell proliferation inhibition assays were performed using the CellTiter-Glo® 2.0 cell viability assay kit (Promega), and cell cycle and apoptosis analyses were performed using a benchtop flow cytometer. Western blot imaging was performed using ChemiDoc MP (Bio-Rad), and band grayscale analysis was performed using ImageJ software. Transcriptome sequencing was performed on the Illumina NovaSeq 6000 platform, differential analysis was performed using DESeq2, and key biological processes and signaling pathways were identified by GO and KEGG enrichment analyses. Animal experiments were conducted using 8-week-old female BALB / c mice.

[0033] (5) Ethics and Compliance All studies strictly adhered to ethical guidelines. In human sample studies, written informed consent was obtained from patients before bone marrow sample collection, the research protocol was approved by the hospital's ethics committee, and the Declaration of Helsinki was followed. Animal experimental protocols were approved by the Animal Ethics Committee of South China University of Technology, and the experimental procedures followed relevant ethical guidelines for animal experiments.

[0034] Example 1: Effects of SGI-7079 on AML cell lines carrying FLT3-ITD mutations in vitro 1. Method (1) Cells and culture conditions AML cell lines carrying FLT3-ITD, MOLM-13 and MV4-11, and wild-type FL-3 control cell lines K562, HL-60, OCI-AML2, OCI-AML3, THP-1, and U937, were selected. Ba / F3 and Ba / F3-FLT3-ITD dependent models were also constructed. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0035] (2) Drug treatment and proliferation detection (CellTiter-Glo) Cells were loaded at 3 × 10 3 SGI-7079, giretinib, and quezatinib were seeded per well in 96-well clear-bottom plates, and treated with different concentrations of each drug. The drugs were prepared using a 3-fold serial dilution method to create a concentration gradient from 0 to 1000 nM, specifically 0 nM, 0.15 nM, 0.46 nM, 1.37 nM, 4.12 nM, 12.35 nM, 37.04 nM, 111.11 nM, 333.33 nM, and 1000 nM, with three replicates per group. After treatment, CellTiter-Glo® 2.0 reagent was added, and the plates were incubated at room temperature for 10 minutes. The signal was measured using a fluorescence / luminescence detector, and the survival rate and dose-response curves were calculated. The IC50 was calculated using nonlinear fitting software. 50 value.

[0036] (3) RNA-seq analysis Total RNA was extracted from MOLM-13 and MV4-11 cells after treatment with 10 nM SGI-7079 for 24 hours. Sequencing libraries were constructed and then sequenced using the Illumina NovaSeq 6000 platform. After quality control, the sequences were aligned to hg38; featureCounts were used for counting. Differential gene expression was analyzed using DESeq2 with a threshold of |log2FC|>1 and FDR<0.05; GO and KEGG enrichment analyses were performed on differentially expressed genes.

[0037] (4) Cell cycle analysis MV4-11 and MOLM-13 cells were mixed at a concentration of 2.0 × 10⁻⁶. 5 Cells were seeded at 100 cells / mL and treated with 3 nM SGI-7079 for 24 hours. Cells were collected, washed twice with PBS, fixed overnight with 70% pre-cooled ethanol, and then incubated at 37°C in the dark for 30 minutes with staining solution containing RNase A (100 μg / mL) and PI (50 μg / mL). The DNA content was determined by flow cytometry.

[0038] (5) Apoptosis detection MV4-11 and MOLM-13 cells were mixed at a concentration of 2.0 × 10⁻⁶. 5 Cells were seeded at 1 / mL in 6-well plates and treated with different concentrations (10, 30, 100, 300 nM) of SGI-7079 for 48 hours. After washing with PBS, the cells were stained according to the Annexin V-FITC / PI kit instructions and incubated at room temperature in the dark for 15 minutes. The apoptosis rate was then detected by flow cytometry.

[0039] (6) Detection of apoptosis-related proteins (Western blot) Cells were treated with different concentrations (10, 30, 100, 300 nM) of SGI-7079 for 48 hours, and total protein (RIPA, containing protease / phosphatase inhibitors) was extracted. After separation by SDS-PAGE, the cells were transferred to a membrane. The cells were incubated with primary antibodies against cleaved Caspase-8 and cleaved PARP1, and detected with HRP-labeled secondary antibody. α-Tubulin was used as an internal control.

[0040] 2. Results Experimental results are as follows Figure 1 As shown, SGI-7079 exhibited a significant inhibitory effect on the proliferation of MOLM-13 and MV4-11 cells, with an IC50 value of [missing information]. 50 The effective doses were 1.93 nM and 0.77 nM, respectively, which were significantly better than giretinib ( Figure 1 (A). It also showed potent inhibitory activity in the Ba / F3-FLT3-ITD cell model. Figure 1 Transcriptome analysis showed that in MV4-11 and MOLM-13 cells treated with SGI-7079, the enrichment of GO and KEGG differentially expressed genes was mainly related to the cell cycle. Figure 1 (C). Flow cytometry further confirmed that SGI-7079 treatment increased the proportion of cells in G1 phase, while decreasing the proportion of cells in S phase and G2 / M phase. Figure 1 (D-E). Apoptosis assays showed that SGI-7079 significantly induced apoptosis in a dose-dependent manner. Figure 1 (F~H). Western blot results further showed that with increasing drug concentration, the expression of cleaved Caspase-8 and cleaved PARP1 significantly increased, confirming the occurrence of apoptosis. Figure 1 (I-J). This indicates that SGI-7079 exhibits potent anti-leukemia activity against AML cell lines carrying the FLT3-ITD mutation in vitro.

[0041] Example 2: Molecular docking, CETSA and downstream signaling pathway verification 1. Method (1) Molecular docking The two-dimensional structure of SGI-7079 was obtained from PubChem, and a three-dimensional conformation was generated using LigPrep. The conformation was then optimized using the OPLS_4 force field to generate protonated states. After processing the FLT3 kinase crystal structure (PDB ID: 6JQR), the ATP pocket was defined as the docking cavity, and docking was performed using the Glide SP mode. The predicted binding affinity was evaluated using GlideScore.

[0042] (2) Cell thermal shift assay (CETSA) 1×10 7 Ba / F3-FLT3-ITD cells were treated with 1 μM SGI-7079 or DMSO for 1 hour. After washing with PBS, the cells were aliquoted into equal parts in PBS containing protease inhibitors, heated in a gradient of 37–55°C for 3 minutes, cooled to room temperature, and lysed in liquid nitrogen. The cells were centrifuged at 20,000 g for 15 minutes, and the supernatant was mixed with 2×SDS loading buffer and heated at 100°C for 15 minutes. Western blot analysis was performed to determine FLT3 protein stability.

[0043] (3) Downstream signal detection (Western blot) After treating MV4-11, MOLM-13 and Ba / F3-FLT3-ITD cells with different concentrations (10, 30, 100, 300 nM) of SGI-7079 for 6 hours, proteins were extracted, and the levels of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK, and ERK were detected.

[0044] 2. Results Experimental results are as follows Figure 2 As shown. Molecular docking reveals that SGI-7079 binds to the ATP pocket of FLT3 DFG-in conformation, forming key hydrogen bonds (Glu692, Cys694), resulting in a stable binding conformation. Figure 2 (A-B). CETSA results showed a significant thermal shift in the FLT3 melting curve, suggesting that the SGI-7079 stable binding protein ( Figure 2 (C-D). Western blot showed decreased levels of p-FLT3 and downstream p-STAT5, p-AKT, and p-ERK. Figure 2 (E-G); RNA-seq KEGG pathway enrichment analysis showed significant enrichment of PI3K / Akt and MAPK signaling pathways (E-G); Figure 2 (H~I). Experimental molecular docking and cellular thermal denaturation analysis (CETSA) confirmed the highly stable binding and targeting effect of SGI-7079 to FLT3. SGI-7079 can achieve high-affinity stable binding by directly targeting the FLT3-ITD protein, thereby overcoming drug resistance caused by FLT3-TKD resistance mutations.

[0045] Example 3: In vivo efficacy of SGI-7079 in a Ba / F3-FLT3-ITD mouse leukemia model 1. Method (1) Establishment of mouse model Eight-week-old female BALB / c mice were intravenously injected with Ba / F3-FLT3-ITD cells (5 × 10⁻⁶). 5(each / animal). Randomly grouped on day 2.

[0046] (2) Dosing regimen SGI-7079 (60 mg / kg, intraperitoneal injection), gidatetinib (30 mg / kg, oral), quezatinib (10 mg / kg, oral), or solvent control, for 8 consecutive days. Regularly weigh and record deaths.

[0047] (3) Detection indicators The proportion of GFP-positive cells in peripheral blood, bone marrow, and spleen was detected by flow cytometry, and spleen weight and H&E histological staining were measured.

[0048] 2. Results Experimental results are as follows Figure 3 As shown, SGI-7079 significantly reduced the proportion of GFP-positive leukemia cells in peripheral blood, bone marrow, and spleen. Figure 3 (B-D), spleen weight decreased ( Figure 3 (E-F), H&E showed a reduction in leukemia infiltration in the liver and spleen after SGI-7079 administration ( Figure 3 (G).

[0049] Example 4: Activity of SGI-7079 on Ba / F3 cell lines carrying secondary drug-resistant TKD mutations 1. Method (1) Construction of drug-resistant cell lines FLT3-ITD plasmids containing different drug resistance mutations (F691L, D835Y / V / F, Y842C, etc.) were transfected into Ba / F3 cells, and drug resistance mutant cell lines were obtained by screening.

[0050] (2) Cell viability detection Drug-resistant cells were seeded at an appropriate density in 96-well plates and treated with gradient concentrations of SGI-7079, gipretinib, and quezatinib for 48 hours. CellTiter-Glo was used to determine cell viability and calculate IC50. 50 .

[0051] (3) Signal path detection Drug-resistant mutant cell lines were treated with different concentrations (10, 30, 100, 300 nM) of SGI-7079 for 6 hours, and the levels of pFLT3, FLT3, pSTAT5, STAT5, pAKT, AKT, pERK and ERK were detected by Western blot.

[0052] 2. Results Experimental results are as follows Figure 4 As shown, SGI-7079 effectively inhibited the proliferation of Ba / F3 cells with various drug-resistant mutations, including D835Y / V / F, Y842C, and F691L, with an IC50 concentration of [missing information].50 Lower than giretinib and quezartinib ( Figure 4 (A-G). Western blot showed decreased levels of p-FLT3 and downstream p-STAT5, p-AKT, and p-ERK. Figure 4 The presence of H-L in the data suggests that SGI-7079 can effectively inhibit FLT3 phosphorylation and downstream signaling pathways.

[0053] Example 5: Effect of SGI-7079 on FLT3-ITD-F691L drug-resistant mouse model 1. Method (1) Model building Ba / F3-FLT3-ITD-F691L cells 5×10 5 BALB / c mice were injected via tail vein and randomly assigned to other groups on day 3.

[0054] (2) Dosing regimen The control group (normal saline), quezatinib (10 mg / kg), giretinib (30 mg / kg), and SGI-7079 (60 mg / kg) were administered for 8 consecutive days, and body weight was recorded.

[0055] (3) Detection indicators The proportion of GFP positivity was detected by flow cytometry in peripheral blood, bone marrow, and spleen. Spleen was weighed and subjected to H&E histological evaluation. Kaplan-Meier curve analysis was used to determine survival.

[0056] 2. Results Experimental results are as follows Figure 5 As shown, SGI-7079 significantly reduced the proportion of GFP-positive leukemia cells in peripheral blood, bone marrow, and spleen. Figure 5 (B-D), spleen weight decreased ( Figure 5 (E-F), H&E staining histological analysis showed that after SGI-7079 treatment, leukemia infiltration in the liver and spleen was significantly reduced, and the tissue structure was basically preserved. Figure 5 (G). The median survival time was extended to 23 days, 16 days in the gipritinib group and 13 days in the quezartinib group. Figure 5 (H).

[0057] Example 6: The role of SGI-7079 in the FLT3-ITD-D835Y drug-resistant mouse model 1. Method Similar to Example 5, the model was established using Ba / F3-FLT3-ITD-D835Y cells, with the same grouping and drug administration regimen.

[0058] 2. Results Experimental results are as follows Figure 6As shown. SGI-7079 reduces the proportion of GFP positivity in peripheral blood, bone marrow, and spleen. Figure 6 (B-D) Spleen weight and volume decreased ( Figure 6 (E-F), H&E showed a decrease in leukemia infiltration ( Figure 6 (G). Kaplan-Meier analysis showed a significant increase in survival (G). Figure 6 (H).

[0059] Example 7: Effect of SGI-7079 on primary cells from FLT3-ITD patients 1. Method (1) Patient sample collection and processing Primary leukemia cells were obtained from bone marrow samples of six FLT3-ITD-positive AML patients using density gradient separation. ITD AML cells refer to cells derived from FLT3 Freshly isolated, unimmortified, and uncultured primary leukemia cells from the bone marrow or peripheral blood of ITD-positive acute myeloid leukemia patients naturally carry FLT3. ITD-driven mutations preserve the true biological characteristics and drug response features in vivo.

[0060] (2) Drug treatment and proliferation detection Primary cells from patients were seeded into 96-well plates and treated with different concentrations of SGI-7079, gipretinib, or quezatinib for 48 h. The drugs were prepared using a 3-fold serial dilution method to create concentration gradients from 0 to 1000 nM (0, 0.15, 0.46, 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, and 1000 nM). CellTiter-Glo was used to detect proliferation / survival and calculate IC50. 50 .

[0061] 2. Results Experimental results are as follows Figure 7 As shown, SGI-7079 exhibited potent inhibitory activity in all patient primary cells, with an IC50 value of [missing value]. 50 Significantly lower than giretinib and quezatinib ( Figure 7 (A through F), the combined results show that it has consistently superior inhibitory effects in primary FLT3-ITD AML cells. Figure 7 (G).

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of protection of the present invention.

Claims

1. The application of SGI-7079 in the preparation of drugs for treating FLT3-mutant acute myeloid leukemia, characterized in that, The structural formula of SGI-7079 is shown in formula (I): The FLT3 mutation is an FLT3-ITD mutation, an FLT3-TKD mutation, or an FLT3-ITD mutation accompanied by TKD resistance.

2. The application according to claim 1, characterized in that, The FLT3-ITD-associated TKD resistance mutation is selected from one or more of the following resistance mutations: D835Y, D835V, D835F, Y842C, or F691L.

3. The application according to claim 2, characterized in that, The FLT3-ITD-associated TKD resistance mutation is selected from D835Y and / or F691L.

4. The application according to claim 1, characterized in that, The drug resistance mutation is a drug resistance mutation against FLT3-TKI.

5. The application according to any one of claims 1 to 4, characterized in that, The drug exerts its therapeutic effect by inhibiting the phosphorylation of STAT5, AKT, and ERK in FLT3 and its downstream signaling pathways.

6. The application according to any one of claims 1 to 4, characterized in that, The drug exerts its therapeutic effect by inhibiting the proliferation of FLT3 mutation-positive leukemia cells, blocking the cell cycle, and inducing apoptosis.

7. The application according to any one of claims 1 to 4, characterized in that, The drug exerts its therapeutic effect by inhibiting the infiltration of leukemia cells in the patient's peripheral blood, bone marrow, and spleen.

8. The application according to any one of claims 1 to 4, characterized in that, The concentration of SGI-7079 in the drug is 1 nM to 1 μM.

9. The application according to any one of claims 1 to 4, characterized in that, The drug also contains a pharmaceutically acceptable carrier or excipient.

10. The application according to any one of claims 1 to 4, characterized in that, The drug is available in oral or injectable form.

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