Application of LPIN1 inhibitor in preparation of medicine for treating FLT3-ITD mutant acute myelogenous leukemia

Abstract

The invention discloses application of an LPIN1 inhibitor in preparation of drugs for treating FLT3-ITD mutant acute myelogenous leukemia, reducing tumor load of a patient with the FLT3-ITD mutant acute myelogenous leukemia, improving the survival rate of the patient with the FLT3-ITD mutant acute myelogenous leukemia and / or reducing the proliferation activity of primary cells of the patient with the FLT3-ITD mutant acute myelogenous leukemia. The LPIN1 inhibitor for inhibiting lipid metabolism related enzyme LPIN1 can significantly induce apoptosis of FLT3-ITD mutant AML cells, has limited influence on non-mutant AML cells, and has mutation specificity. More importantly, the LPIN1 inhibitor (such as propranolol) and the FLT3 inhibitor (such as quinatinib) are combined for use, so that a synergistic anti-leukemia effect can be generated, and a remarkable anti-tumor effect is shown in vitro and in a mouse transplantation tumor model, so that the LPIN1 inhibitor and the FLT3 inhibitor have a good application prospect.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of LPIN1 inhibitors in the preparation of drugs for treating FLT3-ITD mutant acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous and complex malignant tumor of the hematopoietic system. FLT3-ITD mutations account for approximately 20%–30% of all AML cases and are a poor prognostic marker. Although the use of FLT3 inhibitors (such as quezartinib and giglitinib) in recent years has improved the initial response rate for FLT3-ITD-mutant AML patients, overall survival improvement has been limited. Drug resistance and relapse remain major challenges in clinical treatment.

[0003] Current research indicates that single-target therapy is insufficient to overcome the heterogeneity of AML, making combined intervention strategies crucial for overcoming treatment bottlenecks. Against this backdrop, exploring the specific molecular mechanisms of FLT3-ITD mutant AML and identifying novel molecular targets with targeting value have become current research hotspots. Summary of the Invention

[0004] The purpose of this invention is to address the technical deficiencies existing in the prior art. In a first aspect, this invention provides the use of LPIN1 inhibitors in the preparation of drugs for treating FLT3-ITD mutant acute myeloid leukemia, reducing the tumor burden of patients with FLT3-ITD mutant acute myeloid leukemia, improving the survival rate of patients with FLT3-ITD mutant acute myeloid leukemia, and / or reducing the proliferative activity of primary cells in patients with FLT3-ITD mutant acute myeloid leukemia.

[0005] In some embodiments, the LPIN1 inhibitor is a molecule that can inhibit LPIN1 expression or function, including but not limited to: propranolol, propafenone, rosiglitazone, and pharmaceutically acceptable salts or solvates thereof; human LPIN1 siRNA sequences targeting LPIN1 (e.g., siLPIN1-1: 5'-GCAUGAAGUUCUUCGAGAAdTdT-3' (SEQ ID NO:1); siLPIN1-2: 5'-CCACCAAGAUUCUGGACUAdTdT-3' (SEQ ID NO:2)); shRNA vectors targeting LPIN1 (e.g., pLKO.1-shLPIN1, 5'-CCGGGCATGAAGTTCTTCGAGAACTCGAGTTCTCGAAGAACTTCATGCTTTTTG-3' (SEQ ID NO:3)) or CRISPR / Cas9 systems (e.g., including gRNA1: 5'-GAGCCGCTGCGCTACGCCGG-3' (SEQ ID NO:3)). NO:4); and / or gRNA2: 5'-GCTGCGCTACGCCGGCTCCG-3' (SEQ ID NO:5); and / or proteins, small molecule compounds or natural products with LPIN1 inhibitory activity, such as CBM-301106, FSG67, resveratrol, and anti-LPIN1 monoclonal antibodies.

[0006] In some embodiments, the siRNA, shRNA, or CRISPR / Cas9 system targeting LPIN1 is a siRNA, shRNA, or CRISPR / Cas9 system that targets and knocks out or reduces LPIN1.

[0007] In some embodiments, the LPIN1 inhibitor is propranolol or a pharmaceutically acceptable salt or solvate thereof.

[0008] In some embodiments, the FLT3-ITD mutant acute myeloid leukemia has an internal tandem repeat mutation in the FLT3 gene.

[0009] In some embodiments, the FLT3 gene internal tandem repeat mutation is confirmed by molecular detection as a positive FLT3 gene internal tandem repeat mutation with a specific proliferation phenotype.

[0010] In a second aspect, the present invention provides a composition comprising an LPIN1 inhibitor and an FLT3 inhibitor.

[0011] In some embodiments, the LPIN1 inhibitor is a molecule that inhibits the expression or function of LPIN1, optionally including, but not limited to, propranolol or its pharmaceutically acceptable salts or solvates.

[0012] In some embodiments, the LPIN1 inhibitor is a siRNA, shRNA, or CRISPR / Cas9 system that targets and knocks out or reduces LPIN1.

[0013] In some embodiments, the LPIN1 inhibitor is a protein, small molecule compound, or natural product with LPIN1 inhibitory activity.

[0014] In some embodiments, the FLT3 inhibitor is one or more selected from quizartinib, gilteritinib, and sorafenib.

[0015] In some embodiments, the composition is a tablet, capsule, injection, or sustained-release formulation.

[0016] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described composition and a pharmaceutically acceptable carrier or excipient.

[0017] Fourthly, the present invention provides the use of the above-described composition or the above-described pharmaceutical composition in the preparation of a medicament for treating FLT3-ITD mutant acute myeloid leukemia, reducing the tumor burden of patients with FLT3-ITD mutant acute myeloid leukemia, improving the survival rate of patients with FLT3-ITD mutant acute myeloid leukemia, and / or reducing the proliferative activity of primary cells in patients with FLT3-ITD mutant acute myeloid leukemia.

[0018] Fifthly, the present invention provides the use of an LPIN1 inhibitor in combination with an FLT3 inhibitor in the preparation of a medicament for treating FLT3-ITD-mutant acute myeloid leukemia, reducing the tumor burden in patients with FLT3-ITD-mutant acute myeloid leukemia, improving the survival rate of patients with FLT3-ITD-mutant acute myeloid leukemia, and / or reducing the proliferative activity of primary cells in patients with FLT3-ITD-mutant acute myeloid leukemia.

[0019] In some embodiments, the LPIN1 inhibitor and the FLT3 inhibitor may be administered simultaneously or sequentially, and may be repeated over multiple cycles.

[0020] This invention integrates transcriptome (RNA-seq) and translatome (Ribo-seq) sequencing data to perform multidimensional analysis on FLT3-ITD mutant AML samples and non-mutant samples. It is the first time that the lipid metabolism-related enzyme LPIN1 is significantly highly expressed in FLT3-ITD mutant AML and is closely related to cell proliferation and apoptosis.

[0021] Further studies have shown that inhibiting LPIN1 expression or activity can significantly induce apoptosis in FLT3-ITD mutant AML cells, with limited effect on non-mutant AML cells, exhibiting mutation specificity. More importantly, the combined use of LPIN1 inhibitors (such as propranolol) and FLT3 inhibitors (such as quezartinib) can produce a synergistic anti-FLT3-ITD mutant AML effect, demonstrating significant tumor-suppressive effects in both in vitro and mouse xenograft models, suggesting promising application prospects. Attached Figure Description

[0022] Figure 1 The bar chart showing the expression of LPIN1 at the transcriptional and translational levels in FLT3-ITD mutant AML patients compared to non-mutant AML patients in the examples illustrates this.

[0023] Figure 2 This image shows the expression of LPIN1 in a public database in the cell line used in the examples.

[0024] Figure 3 This example shows the proliferation of MOLM13 cells after LPIN1 knockdown by lentivirus in the embodiment.

[0025] Figure 4 The bar charts shown in the examples illustrate the effects of different concentrations of propranolol on the cell viability of AML cell lines; where panel A shows the inhibitory effect of propranolol on HL-60 cells at different time points; panel B shows the inhibitory effect of propranolol on MV4-11 cells at different time points; and panel C shows the inhibitory effect of propranolol on MOLM13 cells at different time points.

[0026] Figure 5 The illustration shows the effect of propranolol on the apoptosis rate of FLT3-ITD mutant AML cell lines at 48 hours in the examples; where A is the effect of propranolol on MOLM13 apoptosis; and B is the effect of propranolol on MV4-11 apoptosis.

[0027] Figure 6 The study demonstrated the killing effect of quezartinib on the LPIN1 knockdown MOLM13 cell line in the assay.

[0028] Figure 7The bar charts shown in the examples illustrate the killing effect of quezartinib combined with propranolol on AML cell lines; wherein, panel A is a bar chart of the killing effect of the two drugs combined on HL-60; panel B is a bar chart of the killing effect of the two drugs combined on MV4-11; and panel C is a bar chart of the killing effect of the two drugs combined on MOLM13.

[0029] Figure 8 The illustration shows a schematic diagram of the experimental process for evaluating the therapeutic effect of LPIN1 inhibitors in an in vivo tumor model of immunodeficient mice.

[0030] Figure 9 The graphs show the therapeutic effects of each group in the mouse tumor model in the examples.

[0031] Figure 10 The graph shows the qualitative and quantitative characterization of tumor burden in the spleen and bone marrow of each group in the mouse tumor model shown in the examples.

[0032] Figure 11 The graph shows the qualitative and quantitative characterization of tumor burden in each group in the mouse tumor model of the example.

[0033] Figure 12 The graphs shown in the examples illustrate the therapeutic effects of each drug treatment on primary cells from three patients (A, B, and C). Detailed Implementation

[0034] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified).

[0035] Example 1: The materials involved in this embodiment include: 1. Mice: Severely immunodeficient mice (NSG) (Beijing Spaford Company).

[0036] 2. Cells: HL-60 cell line; MOLM13 cell line; MV4-11 cell line were all kindly provided by Professor Yu Jia of the Chinese Academy of Medical Sciences.

[0037] 3. Antibody: PIN1 polyclonal antibody was purchased from Thermo Fisher Scientific, USA.

[0038] 4. Main reagents: Quetzartinib was purchased from Selleck, USA; Propranolol was purchased from MCE, USA; RPM1-1640 and fetal bovine serum were purchased from Thermo Fisher Scientific, USA; Cell Counting Kit-8 cell proliferation assay kit was purchased from Nanjing Novizan Biotech Co., Ltd.; Annexin V-APC / 7-AAD cell apoptosis assay kit was purchased from Nanjing KGI Biotech Co., Ltd.

[0039] 5. Major instruments: Flow cytometer purchased from BD Biosciences, USA; Microplate reader purchased from PerkinElmer, USA.

[0040] Experiment 1: Analysis of RNA-seq and Ribo-seq data In this experiment, the md5sum command was used to check the integrity of RNA-seq and Ribo-Seq data (patient samples were from the Fifth Medical Center of the General Hospital of the Chinese People's Liberation Army, and sequencing was performed by Novogene). Cutadapt software (v1.14) was used to remove adapter sequences and filter low-quality sequences, and Bowtie2 (version 2.2.5) was applied to remove rRNA, tRNA, and mitochondrial (MT) sequences. To eliminate fluctuations in the first few bases of each read sequence caused by instability at the beginning of the synthesis reaction of the sequencer, the first 3 bases of the read were removed, and the preprocessed RNA-seq and Ribo-seq reads were aligned to the human genome GRCh38 (GRCh38 Release 36, https: / / www.gencodegenes.org / human / release_36.html) using STAR v2.7.6a (parameters set as: --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFilterMismatchNmax 2 --outSAMattributes All --outSAMtype BAM Unsorted --outFilterType BySJout).

[0041] The BAM files were processed using SAMtools software (v0.1.19-44428cd), and uniquely aligned sequences were selected, sorted, and indexed. PCR duplicates were then removed using Python and Perl scripts, and the results were again sorted and indexed using SAMtools software (v0.1.19-44428cd) to obtain the final BAM files. FastQC software (v0.11.5) was used to check the quality of the raw data and examine data characteristics to ensure that the average quality score of each sequence met the required standard.

[0042] The sequences were aligned to the human reference genome (Ensembl GRCh38release-83, http: / / ftp.ensembl.org / pub / release-83 / fasta / homo_sapiens / dna / ) using STAR software (v2.7.3a) to obtain unique aligned sequences. After determining strand specificity, the counts corresponding to the exon regions of the genome for each sample were calculated using featureCounts software (v1.6.3), and the counts file was obtained. The FPKM (Fragments Per Kilobase Per Million mapped fragments) of all sample genes was also calculated. The results are shown in [link to results]. Figure 1 .

[0043] Figure 1 The results showed that in patients with FLT3-ITD mutant AML, the expression of LPIN1, a gene involved in lipid metabolism, was significantly higher at both the transcriptional and translational levels than in patients with non-mutant AML.

[0044] Experiment 2: LPIN1 knockdown and drug intervention in AML cell lines First, the expression of LPIN1 in various AML cell lines (such as HL-60, MV4-11, MOLM13, THP-1, and U937) was compared in the CCLE database (https: / / sites.broadinstitute.org / ccle / ) (see [link to CCLE database](https: / / sites.broadinstitute.org / ccle / )). Figure 2 Ultimately, HL-60 cell lines with low LPIN1 expression and no FLT3-ITD mutation were selected as the control group, while MOLM13 and MV4-11 cell lines with high LPIN1 expression and FLT3-ITD mutation were selected as the experimental groups for subsequent cell experiments.

[0045] Next, the LPIN1 gene was knocked down in the FLT3-ITD mutant AML cell line MOLM13 using a lentiviral vector (purchased from Shanghai Kaiji Gene Co., Ltd., Hu6-MSC-CMV-Puromycin), constructing an LPIN1 knockdown cell line (MOLM13-LPIN1 KD). Specifically, the virus was packaged and co-transfected into 293T cells with psPAX2 and pMD2.G for 48 hours. The supernatant containing viral particles was collected to infect the target MOLM13 cells. After 24 hours, the cells were screened using complete medium containing 1 μg / mL puromycin until a stable LPIN1 knockdown cell line was obtained. At the same time, an empty vector control group (MOLM13-vector) was constructed.

[0046] To assess the effect of LPIN1 knockdown on the proliferation of MOLM13 cells, cell viability / proliferation was detected using the Cell Counting Kit-8. The simplified procedure is as follows: MOLM13-vector and MOLM13-LPIN1 KD cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL in 96-well plates and pre-cultured at 37°C in a 5% CO2 incubator. After 24, 48, 72, and 96 hours of pre-culture, 10 µL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours in the dark. Finally, the absorbance of each well was measured at 450 nm using a multi-mode microplate reader. The relative percentage of cell viability (also known as "cell viability percentage") was calculated using the following formula: Cell viability (%) = [(Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value)] × 100%. All experiments were independently repeated at least three times. Results are shown in the table below. Figure 3 .

[0047] Figure 3 The results showed that, compared with control cells (MOLM13-vector), knocking down LPIN1 significantly inhibited the proliferation of MOLM13 cells, suggesting that inhibiting LPIN1 expression may inhibit the proliferation of the FLT3-ITD mutant cell line MOLM13.

[0048] Furthermore, HL60, MOLM13, and MV4-11 cells were seeded into 6-well plates, with 2 × 10⁶ cells per well. 5 Cells were treated with DMSO solution of propranolol for 24 hours, at concentration gradients of 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM. A DMSO solvent control group was also included. Treatment times were 24, 48, and 72 hours. Samples were taken and analyzed, and the percentage of cell viability was calculated using the method described above. Results are shown below. Figure 4 .

[0049] Figure 4 The results showed that propranolol could inhibit the proliferation of HL-60 cells, MV4-11 cells, and MOLM13 cells in a dose-dependent manner, but the IC50 value in these three cell types was significantly lower. 50 There are significant differences. Taking 48 h as an example, the IC50 values ​​of the three cell lines... 50 The concentrations were 137.3 μmol / L, 106.3 μmol / L, and 93.8 μmol / L, respectively, indicating that cells with high LPIN1 expression were more sensitive to inhibitors.

[0050] The apoptosis rate of propranolol-treated MOLM13 and MV4-11 cells was further detected by Annexin V-FITC / PI double staining flow cytometry. Cells treated with the drug were collected, washed once with PBS, resuspended in binding buffer, and then 5 μL of Annexin V-FITC and 5 μL of PI were added. After incubation in the dark for 15 minutes, the cells were analyzed. Data analysis was performed using FlowJo software. The apoptosis rate was calculated as the sum of the Annexin V+ / PI− and Annexin V+ / PI+ cell ratios. Results are shown in [Figure number missing]. Figure 5 .

[0051] Figure 5 The results showed that propranolol could significantly induce apoptosis in FLT3-ITD mutant AML tumor cells, demonstrating potential anti-tumor effects (P<0.01).

[0052] Experiment 3: In vitro synergistic experiment of LPIN1 inhibitor combined with FLT3 inhibitor MOLM13 cells with LPIN1 knockdown (referred to as "MOLM13 / LPIN1 knockdown") and those without LPIN1 knockdown (referred to as "MOLM13 / blank knockdown") were treated with 100 μM quinzatinib (as an FLT3 inhibitor) for 24 h, 48 h, and 72 h, respectively. Cell viability was assessed using the CCK-8 assay. Specifically, 10 μL CCK-8 reagent was added to each well, incubated for 1 h, and absorbance was measured at 450 nm. The percentage of cell viability was calculated following the procedure in Experiment II. The results are shown in [Figure 1]. Figure 6 .

[0053] The results showed that, compared with control cells (MOLM13 / blank gene knockdown), knockdown of LPIN1 significantly inhibited cell proliferation (see [link to study]). Figure 6 ).

[0054] Furthermore, HL-60, MV4-11, and MOLM13 cells were treated with 100 μM propranolol (as an LPIN1 inhibitor) in combination with 5 nM quezartinib. Single-drug control and combination groups were established. Cell viability percentage was assessed using the CCK-8 assay after 24, 48, and 72 hours of treatment. A significantly higher inhibition rate in the combination group compared to any single-drug group indicated a synergistic effect. The specific method was the same as above; the results at 48 hours are shown in the table below. Figure 7 .

[0055] Figure 7 The results showed that knocking down LPIN1 significantly increased the sensitivity of MOLM13 to quezartinib, indicating that LPIN1 plays an important role in the growth and proliferation of this type of AML cells. Furthermore, taking 48 h as an example, in MV4-11 cells, propranolol combined with quezartinib reduced the IC50 of quezartinib. 50 The concentration of propranolol decreased from 0.639 nmol / L to 0.2424 nmol / L; in MOLM13 cells, propranolol in combination with quezartinib reduced the IC50 of quinzatinib. 50 The concentration decreased from 1.613 nmol / L to 0.5308 nmol / L. However, in HL-60 cells with low LPIN1 expression, propranolol combined with quezartinib showed an IC50 reduction. 50 The reduction was not significant. The above experiments indicate that propranolol in combination with quezartinib can significantly reduce the IC50 of quezartinib. 50 This indicates that inhibiting LPIN1 expression through lentiviral knockdown or intervention with specific inhibitors can enhance the targeted killing effect of quezatinib on FLT3-ITD mutant AML.

[0056] Experiment 4: Construction of an AML xenograft model and drug intervention A schematic diagram of the experimental process can be found here. Figure 8 MOLM13 cells (5 × 10⁻⁶) 6 (Number of mice) were injected via the tail vein into severely immunodeficient mice, followed by daily injections of placebo (i.e., solvent), propranolol (10 mg / kg), quezartinib (10 mg / kg), and propranolol (10 mg / kg) combined with quezartinib (10 mg / kg) for four weeks. Each group consisted of 13 mice; 8 were used for survival analysis and 5 for disease progression analysis.

[0057] Survival analysis specifically involved observing the survival status of mice; the results are shown below. Figure 9 .

[0058] Figure 9 The results showed that more mice in the two-drug combination group achieved longer survival days.

[0059] The specific steps for disease progression analysis are as follows: After treatment, mice were euthanized, and their spleens, bilateral femurs (with complete dissection of surrounding muscle tissue), and retroorbital blood samples were collected. The obtained spleen and femur tissue samples were immediately and completely immersed in a sufficient amount of 4% paraformaldehyde phosphate buffer fixative and fixed at 4°C for 24-48 hours. After fixation, the tissues were rinsed three times with phosphate-buffered saline (PBS) to remove the fixative. Subsequently, the femur tissue was transferred to 10% EDTA decalcification buffer (pH 7.4) and gently decalcified at room temperature on a shaker. The decalcification process lasted 1 to 2 weeks, during which the decalcification solution was changed daily until no resistance was felt when puncturing the femur tissue with a fine needle, indicating complete decalcification.

[0060] Fixed spleen tissue (after removal of fixative) and decalcified femoral tissue were sequentially dehydrated using a gradient of ethanol (70%, 80%, 90%, 95%, 100%), with each gradient lasting 1-2 hours. The dehydrated tissues were then cleared with xylene to replace the ethanol. Subsequently, the tissues were immersed in molten paraffin for embedding, creating tissue blocks. The paraffin blocks were serially sectioned using a paraffin microtome to a thickness of 4 μm. The flattened sections were then retrieved and attached to poly-L-lysine-pretreated glass slides. The slides with the sections were placed in a 60°C oven for at least 1 hour to ensure tight adhesion and evaporate any residual moisture.

[0061] Before staining, paraffin sections need to be dewaxed and hydrated. The specific steps are as follows: First, immerse the sections in xylene I for 10 minutes to dissolve the paraffin, then transfer them to xylene II for 10 minutes to ensure complete dewaxing. Next, hydrate the sections sequentially in ethanol solutions of decreasing concentration: 100% ethanol for 5 minutes, 95% ethanol for 3 minutes, 85% ethanol for 3 minutes, and 75% ethanol for 3 minutes. Finally, rinse the sections twice in running deionized water or phosphate-buffered saline (PBS), 5 minutes each time, to complete the "to-water" process and restore the tissue to a state suitable for aqueous staining.

[0062] Following this, hematoxylin-eosin (H&E) staining is performed: the sections are immersed in Harris hematoxylin staining solution for 5 minutes to stain the cell nuclei. After removal, they are extracted in 1% hydrochloric acid ethanol differentiation solution for a few seconds to remove non-specific staining of the cytoplasm, and immediately rinsed slowly with running tap water for at least 15 minutes to fully reveal the blue color of the cell nuclei (blue reversion). Next, the sections are counterstained in 0.5% eosin Y aqueous solution or alcohol solution for 2 minutes to stain the cytoplasm and connective tissue pink. After staining, the sections need to be dehydrated stepwise with a gradient of ethanol (75%, 85%, 95%, 100%), soaking for 2-3 minutes at each stage. After dehydration, they are cleared sequentially with xylene I and xylene II, soaking for 5-10 minutes at each stage. Finally, an appropriate amount of neutral resin is dropped onto the tissue area, and a coverslip is carefully placed over it to avoid air bubbles. The slide is then allowed to cure at room temperature in the dark, completing the preparation for subsequent microscopic observation and image acquisition and analysis.

[0063] To detect LPIN1 protein expression, additional slides were prepared, treated with antigen retrieval (sodium citrate buffer, pH 6.0, heated at 95°C for 20 minutes), followed by endogenous peroxidase blockade with 3% H2O2 for 10 minutes and blocking with 10% normal goat serum for 30 minutes. Subsequently, the slides were incubated with primary antibody (rabbit anti-LPIN1, incubated overnight at 4°C), secondary antibody (HRP-labeled goat anti-rabbit IgG, incubated at room temperature for 30 minutes), DAB staining for 3 minutes, hematoxylin counterstaining for 1 minute, and finally dehydrated, cleared, and mounted. All slides were observed and images were acquired under an optical microscope (see [link to image]). Figure 10 (Left side of the image). CD45-positive cell membrane markers were used as surface markers for acute myeloid leukemia (AML) cells to locate and quantify AML cell infiltration in bone marrow or spleen tissues. Simultaneously, the mean optical density of LPIN1 protein expression in target regions (such as CD45+ cell regions or specific tissue regions) was analyzed using ImageJ software to quantify its expression level. Data are expressed as mean ± standard deviation. Statistical analysis was performed using t-tests for intergroup comparisons. P < 0.05 was considered statistically significant. Results are shown in [link to results]. Figure 10 The right side of the image.

[0064] Add approximately 1–2 mL of ACK lysis buffer to a blood sample collected from the retro-orbital region of mice, and gently pipette to mix. Incubate at room temperature for 5 minutes to lyse red blood cells. Neutralize with PBS, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in FACS buffer. Add anti-human CD45 fluorescent antibody, incubate on ice or at 4°C in the dark for 30 minutes, wash with 1–2 mL of FACS buffer, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and resuspend in 200 µL of FACS buffer for analysis. Remove impurities and debris using FSC / SSC gating; detect the percentage or absolute count of CD45-positive cells. Results are shown in [Figure number missing]. Figure 11 .

[0065] Figure 10 and Figure 11 The results showed that, compared with the control group, both propranolol and quezartinib monotherapy significantly inhibited tumor growth, suggesting that both have certain in vivo anti-leukemia activity. However, the tumor inhibition effect was more significant in the combination therapy group, with tumor volume and leukemia cell infiltration levels almost reduced to undetectable levels. Quantitative analysis showed that propranolol and quezartinib monotherapy reduced tumor burden by approximately 70-80%, while the inhibition rate of the combination therapy exceeded 95%. Histological examination and flow cytometry further confirmed that leukemia cells in the bone marrow and spleen of mice in the combination therapy group were significantly reduced. Statistical analysis showed that the tumor inhibition effect of the combination therapy group was significantly higher than that of either monotherapy group (p<0.0001). In conclusion, propranolol can significantly enhance the anti-leukemia activity of quezartinib in vivo, and the combination of the two drugs produced a synergistic therapeutic effect.

[0066] Experiment 5: Primary Cell Extraction and Drug Intervention Experiment from Patients Bone marrow samples were collected from AML patients with informed consent. Mononuclear cells (BM-MNCs, as primary cells) from the three patients were isolated using Ficoll density gradient centrifugation and labeled as groups A, B, and C. After washing with PBS, the cells were counted and used in subsequent experiments. BM-MNCs were seeded in 96-well plates, 1 × 10⁶ cells per well. 5 Cells were supplemented with RPMI-1640 medium containing 10% FBS and divided into quinzartinib monotherapy and dual-drug combination therapy groups. Cell proliferation was assessed using a microplate reader after 24 and 48 hours of treatment, and cell viability percentage was used for characterization. Specific detection and calculation methods are described in Experiments II and III. Results are shown below. Figure 12 .

[0067] Figure 12 The results showed that the proliferation activity of primary cells from patients with FLT3-ITD mutations was significantly lower in the combined drug treatment group than in the quezartinib monotherapy group, further supporting the specific targeting value of LPIN1 in FLT3-ITD mutant AML.

[0068] All the above experimental results indicate that LPIN1 knockdown can significantly increase the sensitivity of FLT3-ITD mutant AML cells to quezartinib; and the combination therapy of propranolol and quezartinib showed synergistic antitumor effects in vitro and in mouse models, indicating that LPIN1 has potential intervention target value in FLT3-ITD AML and provides a new strategy for targeted therapy of AML.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered as part of the present invention.

Claims

1. The use of LPIN1 inhibitors in the preparation of drugs for the treatment of FLT3-ITD mutant acute myeloid leukemia, reducing tumor burden in patients with FLT3-ITD mutant acute myeloid leukemia, improving survival rate in patients with FLT3-ITD mutant acute myeloid leukemia, and / or reducing the proliferative activity of primary cells in patients with FLT3-ITD mutant acute myeloid leukemia.

2. The application according to claim 1, characterized in that, The LPIN1 inhibitor is a molecule that can inhibit LPIN1 expression or function, including but not limited to: propranolol, propafenone, rosiglitazone, and their pharmaceutically acceptable salts or solvates; human LPIN1 siRNA sequences targeting LPIN1 (e.g., siLPIN1-1: 5'-GCAUGAAGUUCUUCGAGAAdTdT-3'; or siLPIN1-2: 5'-CCACCAAGAUUCUGGACUAdTdT-3'); shRNA vectors targeting LPIN1 (e.g., pLKO.1-shLPIN1, 5'-CCGGGCATGAAGTTCTTCGAGAACTCGAGTTCTCGAAGAACTTCATGCTTTTTG-3') or CRISPR / Cas9 systems (e.g., including gRNA1: 5'-GAGCCGCTGCGCTACGCCGG-3'; and / or gRNA2: 5'-GCTGCGCTACGCCGGCTCCG-3'); and / or proteins, small molecule compounds or natural products with LPIN1 inhibitory activity, such as CBM-301106, FSG67, resveratrol, and / or anti-LPIN1 monoclonal antibodies; Preferably, the siRNA, shRNA, or CRISPR / Cas9 system targeting LPIN1 is a siRNA, shRNA, or CRISPR / Cas9 system that targets and knocks out or reduces LPIN1. More preferably, the LPIN1 inhibitor is propranolol or a pharmaceutically acceptable salt or solvate thereof.

3. The application according to claim 1 or 2, characterized in that, The FLT3-ITD mutant acute myeloid leukemia has an internal tandem repeat mutation in the FLT3 gene; preferably, the internal tandem repeat mutation in the FLT3 gene is confirmed by molecular detection as a positive internal tandem repeat mutation in the FLT3 gene and has a specific proliferative phenotype.

4. A composition, characterized in that, Including LPIN1 inhibitors and FLT3 inhibitors.

5. The composition according to claim 4, characterized in that, The LPIN1 inhibitor is a molecule that can inhibit the expression or function of LPIN1, and optionally includes, but is not limited to, propranolol or its pharmaceutically acceptable salts or solvates; and / or The LPIN1 inhibitor is a siRNA, shRNA, or CRISPR / Cas9 system that targets and knocks out or reduces LPIN1; and / or The LPIN1 inhibitor is a protein, small molecule compound, or natural product with LPIN1 inhibitory activity.

6. The composition according to claim 4 or 5, characterized in that, The FLT3 inhibitor is selected from one or more of quizartinib, gilteritinib, and sorafenib.

7. The composition according to any one of claims 4-6, characterized in that, It is available in tablet, capsule, injection, or sustained-release formulations.

8. A pharmaceutical composition comprising the composition of any one of claims 4-7 and a pharmaceutically acceptable carrier or excipient.

9. The use of the composition of any one of claims 4-7 or the pharmaceutical composition of claim 8 in the preparation of a medicament for treating FLT3-ITD mutant acute myeloid leukemia, reducing tumor burden in patients with FLT3-ITD mutant acute myeloid leukemia, improving survival rate in patients with FLT3-ITD mutant acute myeloid leukemia, and / or reducing the proliferative activity of primary cells in patients with FLT3-ITD mutant acute myeloid leukemia.

10. The use of LPIN1 inhibitors in combination with FLT3 inhibitors in the preparation of drugs for the treatment of FLT3-ITD mutant acute myeloid leukemia, reducing tumor burden in patients with FLT3-ITD mutant acute myeloid leukemia, improving survival rate in patients with FLT3-ITD mutant acute myeloid leukemia, and / or reducing the proliferative activity of primary cells in patients with FLT3-ITD mutant acute myeloid leukemia.

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