Use of acsl4 inhibitors in the manufacture of a medicament for treating acute myeloid leukemia

Abstract

This application belongs to the field of biomedical technology, specifically relating to the use of an ACSL4 inhibitor in the preparation of a drug for treating acute myeloid leukemia (AML). This application discloses for the first time the novel use of an ACSL4 inhibitor in the preparation of a drug for treating AML. In vitro experiments show that the ACSL4 inhibitor can effectively inhibit AML cell proliferation and promote apoptosis, without significantly affecting peripheral blood mononuclear cells in healthy controls. Animal experiments further confirm that the ACSL4 inhibitor can significantly delay disease progression in IGF2BP3-overexpressing AML mice, reduce tumor burden, and prolong the survival of NCG mice, further confirming its anti-leukemic effect in vivo.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to the use of an ACSL4 inhibitor in the preparation of a drug for treating acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a type of hematologic malignancy characterized by abnormal proliferation, differentiation disorders, and apoptosis arrest of myeloid hematopoietic stem / progenitor cells. It accounts for approximately 80% of adult acute leukemia cases and is the most common type of adult acute leukemia clinically. The development and progression of AML are closely related to the abnormal regulation of multiple molecular pathways. Its core pathological changes lead to impaired normal hematopoietic function, and patients often present with a series of clinical symptoms such as anemia, bleeding, and infection. Although medical technology has made continuous progress in recent years, and breakthroughs have been achieved in the diagnosis and treatment of AML, the high heterogeneity and complex molecular mechanisms of the disease make it a challenge for clinical treatment, resulting in a generally poor prognosis for patients. There is an urgent need to explore new pathogenesis and treatment strategies to improve clinical outcomes.

[0003] Currently, AML treatment options mainly include traditional chemotherapy, hematopoietic stem cell transplantation, and targeted therapy targeting specific molecular targets. Traditional chemotherapy works by broadly killing rapidly proliferating cells, and while it can achieve complete remission in some patients, it suffers from drawbacks such as poor specificity and severe damage to normal hematopoietic cells, leading to a high incidence of treatment-related adverse reactions and poor tolerability. Approved targeted drugs, such as FLT3 inhibitors and IDH inhibitors, have shown some efficacy in certain subtypes of AML, but are limited by narrow target coverage and exhibit primary or secondary drug resistance in some patients. Furthermore, the development of novel targeted drugs often requires lengthy basic research, preclinical trials, and clinical trials, resulting in long development cycles and high costs, significantly increasing the treatment burden on patients. In addition, the pathogenesis of AML is closely related to several novel regulatory factors. Previous studies have found abnormally high expression of IGF2BP3 in AML patients, and combined with multi-omics sequencing results and mechanistic experiments, it has been discovered that the novel IGF2BP3 / UBR7 / ACSL4 axis regulates the occurrence and development of AML. However, there is currently a lack of effective interventions targeting the regulatory pathways mediated by IGF2BP3 overexpression, and treatment options remain very limited for this segment of AML patients with high IGF2BP3 expression.

[0004] Therefore, it is necessary to improve one or more of the problems existing in the above-mentioned related technical solutions.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this disclosure is to provide the use of an ACSL4 inhibitor in the preparation of a medicament for treating acute myeloid leukemia, thereby overcoming at least to some extent one or more problems caused by limitations and defects in related technologies.

[0007] This application provides the use of an ACSL4 inhibitor in the preparation of a medicament for treating acute myeloid leukemia.

[0008] In one possible implementation, the ACSL4 inhibitor targets the ACSL4 protein, inhibiting the proliferation of acute myeloid leukemia cells and promoting their apoptosis.

[0009] In one possible implementation, the ACSL4 inhibitor is at least one of rosiglitazone and PRGL493.

[0010] In one possible implementation, the drug further comprises pharmaceutically acceptable excipients.

[0011] In one possible implementation, the pharmaceutically acceptable excipient is a diluent, binder, surfactant, humectant, adsorbent, lubricant, and / or disintegrant.

[0012] In one possible implementation, the dosage form of the drug is a solution, sustained-release agent, suspension, granules, tablets, capsules, powder, emulsion, syrup, or drops.

[0013] The technical solution provided in this application may include the following beneficial effects: This application discloses for the first time a novel use of ACSL4 inhibitors in the preparation of drugs for the treatment of acute myeloid leukemia (AML). In vitro experiments confirmed that ACSL4 inhibitors inhibit AML cell proliferation and promote AML cell apoptosis, without significantly affecting peripheral blood mononuclear cells in healthy controls. Animal experiments further demonstrated that ACSL4 inhibitors can delay disease progression in IGF2BP3-overexpressing AML mice, reduce tumor burden in mice, and prolong the survival of NCG mice, further confirming their in vivo antileukemic effect.

[0014] Meanwhile, rosiglitazone, an ACSL4 inhibitor, is a thiazolidinedione insulin sensitizer commonly used clinically to treat type 2 diabetes. It is safe, effective, and inexpensive. Compared to expensive chemotherapy drugs and targeted therapies, rosiglitazone can significantly shorten drug development cycles and costs, and substantially improve drug safety in the treatment of AML.

[0015] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Attached Figure Description

[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It is obvious that the drawings described below are merely some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 This illustration shows the expression of ACSL4 protein in peripheral blood mononuclear cells from healthy donors and bone marrow mononuclear cells from newly diagnosed AML patients in exemplary embodiments of this disclosure; Figure 2 This diagram illustrates the proliferation of AML cells after overexpression of ACSL4 in an exemplary embodiment of this disclosure. Figure 3 This diagram illustrates the proliferation of AML cells after ACSL4 knockdown in an exemplary embodiment of this disclosure. Figure 4 This diagram illustrates the cell proliferation of an IGF2BP3-overexpressing AML cell line treated with rosiglitazone in an exemplary embodiment of this disclosure. Figure 5 This diagram illustrates the cell proliferation of an IGF2BP3-overexpressing AML cell line under the action of PRGL493 in an exemplary embodiment of this disclosure. Figure 6 This diagram illustrates the cell viability of primary AML cells treated with rosiglitazone in an exemplary embodiment of this disclosure. Figure 7 This diagram illustrates the cell viability of normal bone marrow mononuclear cells under the action of rosiglitazone in an exemplary embodiment of this disclosure. Figure 8 This diagram illustrates the apoptosis of AML cells after overexpression of ACSL4 in an exemplary embodiment of this disclosure. Figure 9 This diagram illustrates the apoptosis of AML cells after ACSL4 knockdown in an exemplary embodiment of this disclosure. Figure 10 This diagram illustrates the apoptosis of primary AML cells under the action of rosiglitazone in an exemplary embodiment of this disclosure. Figure 11 This diagram illustrates the apoptosis of normal bone marrow mononuclear cells under the action of rosiglitazone in an exemplary embodiment of this disclosure. Figure 12This diagram illustrates the flow cytometry detection of ACSL4 protein expression in primary AML cells after treatment with rosiglitazone, according to an exemplary embodiment of this disclosure. Figure 13 This diagram illustrates the Western Blot detection of ACSL4 protein expression in primary AML cells after treatment with rosiglitazone, according to an exemplary embodiment of this disclosure. Figure 14 This diagram illustrates the total survival time of mice in different drug administration groups in an exemplary embodiment of this disclosure. Figure 15 This diagram illustrates the clinical scores of rosiglitazone acting on IGF2BP3-overexpressing and control AML mice in an exemplary embodiment of this disclosure. Figure 16 This diagram illustrates in vivo imaging of mice in different drug administration groups in an exemplary embodiment of this disclosure. Figure 17 This diagram illustrates the quantitative results of mouse in vivo imaging in an exemplary embodiment of this disclosure. Figure 18 This diagram illustrates the changes in the survival time of NCG mice after using an ACSL4 inhibitor in an exemplary embodiment of this disclosure. Detailed Implementation

[0018] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, they are provided so that this disclosure will be more comprehensive and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0019] This application reveals that the ACSL4 gene and protein can serve as a novel therapeutic target for AML. ACSL4 consists of five regions: an NH2 terminus, luciferase-like regions 1 and 2, a linker connecting these two luciferase-like regions, and a COOH terminus. ACSL4 is mainly distributed in the endoplasmic reticulum, peroxisomes, and mitochondrial-associated membranes. ACSL4 catalyzes the formation of acyl-CoA by inserting CoA into polyunsaturated fatty acids (such as arachidonic acid (AA) and adrenaline (AdA)). These fatty acyl-CoA molecules provide energy to cells through β-FA oxidation or enter lipid biosynthesis pathways to produce AA-containing triacylglycerols (TG), phospholipids (PL), and cholesterol esters (CE). ACSL4 is highly expressed in various cancers, including breast cancer, colon cancer, and liver cancer, and is associated with tumor occurrence, development, and metastasis.

[0020] This example embodiment first provides the use of an ACSL4 inhibitor in the preparation of a medicament for treating acute myeloid leukemia.

[0021] It should be noted that this application uses RNA interference technology to interfere with the abnormal expression of ACSL4 in AML cells. CCK-8 assays showed that overexpression of ACSL4 promoted AML cell proliferation, while knockdown of ACSL4 inhibited cell proliferation. Flow cytometry analysis revealed that overexpression of ACSL4 inhibited AML cell apoptosis, while knockdown of ACSL4 promoted apoptosis. Knockdown of ACSL4 prolonged the overall survival of NCG mice. Rosiglitazone was applied to IGF2BP3-overexpressing AML cell lines, and rosiglitazone inhibited cell proliferation in a time- and concentration-dependent manner. Rosiglitazone was applied to bone marrow mononuclear cells from newly diagnosed AML patients, and rosiglitazone inhibited cell proliferation and promoted apoptosis in a concentration-dependent manner, with no significant effect on peripheral blood mononuclear cells of healthy controls. Flow cytometry and Western blotting confirmed a concentration-dependent decrease in ACSL4 protein expression, indicating that rosiglitazone acts on AML cells through the ACSL4 protein. To further clarify the in vivo anti-leukemic effect of rosiglitazone, we treated IGF2BP3-overexpressing and control mice with rosiglitazone. The results showed that rosiglitazone could delay the onset of AML in IGF2BP3-overexpressing mice, reduce tumor burden, and prolong survival. This experiment demonstrates that ACSL4 is a highly promising therapeutic target for anti-AML, and ACSL4 inhibitors possess significant potential for in vivo anti-leukemic activity.

[0022] It is also important to clarify that the ACSL4 target is specifically highly expressed in acute myeloid leukemia cells. Therefore, when this inhibitor exerts its anti-leukemic effect, it will not significantly interfere with the proliferation of normal hematopoietic cells, which lays a safety foundation for its subsequent clinical translation. Furthermore, proposing this application also provides a new targeted therapy direction for patients with refractory acute myeloid leukemia who are resistant to existing chemotherapy regimens.

[0023] In one embodiment, the ACSL4 inhibitor targets the ACSL4 protein, inhibiting AML cell proliferation and promoting apoptosis.

[0024] It should be noted that the pro-apoptotic effect of ACSL4 inhibitors on acute myeloid leukemia cells is targeted and does not significantly interfere with the physiological activity of normal hematopoietic cells.

[0025] In one embodiment, the ACSL4 inhibitor is at least one of rosiglitazone and PRGL493.

[0026] It should be noted that rosiglitazone is a clinically approved drug for the treatment of type 2 diabetes, and its pharmacokinetic characteristics, dosing regimen, and long-term safety data are well understood; PRGL493 is an ACSL4-specific targeted inhibitor, and both can effectively inhibit ACSL4 when used alone.

[0027] In one embodiment, the drug further comprises pharmaceutically acceptable excipients.

[0028] It should be noted that the pharmaceutically acceptable excipients mentioned here must meet the quality standards of the current national pharmacopoeia, and their addition will not affect the efficacy and safety of ACSL4 inhibitors.

[0029] Furthermore, the pharmaceutically acceptable excipients are diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants.

[0030] It should be noted that different types of excipients need to be selected and combined according to the formulation requirements of the drug. For example, diluents and disintegrants are often used in combination in tablet preparation to ensure the formulation's shape and dissolution.

[0031] In one embodiment, the dosage form of the drug is a solution, sustained-release agent, suspension, granules, tablets, capsules, powder, emulsion, syrup, or drops.

[0032] It should be noted that different dosage forms are designed to meet different clinical needs. For example, sustained-release formulations can prolong the duration of drug effect, while drops are more convenient for children or patients with swallowing difficulties to administer the medication.

[0033] The following is a further explanation and description of the content of this application based on specific embodiments: Experimental materials: The following reagents were used: apoptosis antibody Annexin V-APC and 7-AAD, CCK-8 reagent, rosiglitazone, PRGL493, antibody for Western blotting analysis, and lymphocyte separation medium for isolating bone marrow mononuclear cells. Annexin V-APC and 7-AAD were purchased from BioLegend; CCK-8 reagent from Apebio; rosiglitazone from AbMole; PRGL493 from Medchemexpress; antibody for Western blotting analysis from Abcam and Proteintech; and lymphocyte separation medium for isolating bone marrow mononuclear cells from Biosharp.

[0034] Experimental methods: This application employs four methods for analysis: CCK-8 assay for cell proliferation, flow cytometry analysis of cell apoptosis, Western blotting, flow cytometry analysis of ACSL4 expression, and construction of an intravenous injection animal model.

[0035] Example 1 Western blotting analysis of ACSL4 protein expression was performed as follows: Peripheral blood from healthy controls and bone marrow fluid from newly diagnosed AML patients were collected, and mononuclear cells were extracted. An appropriate amount of RIPA strong lysis buffer (Beyotime) was added according to the cell volume ratio, along with 1× final concentrations of phosphatase inhibitor and protease inhibitor, and the mixture was gently pipetted to mix. The cells were then sonicated on ice (7 seconds on, 8 seconds off, total 35 seconds), followed by 15 minutes of standing on ice to allow for complete lysis. The cells were centrifuged at 13,000 rpm for 15 minutes at 4°C to separate soluble total protein. The supernatant was carefully aspirated into a new EP tube, avoiding aspiration of precipitate. Protein concentration was determined using the Bradford method, and a standard curve was constructed using BSA for quantification. The volume V of the protein sample was measured. 样品 According to formula V buffer =V 样品 / 4 Calculate the volume of 5× protein buffer to be added for each sample; add 5× protein buffer and mix well (final concentration 1×); after denaturing at 99℃ for 10 minutes, calculate the loading volume according to the required loading amount and perform SDS-PAGE electrophoresis.

[0036] Analysis results: such as Figure 1 The image shows the expression of ACSL4 protein in peripheral blood mononuclear cells from healthy controls and in bone marrow mononuclear cells from newly diagnosed AML patients. The results showed that ACSL4 protein expression in bone marrow mononuclear cells from newly diagnosed AML patients was higher than that in the control group.

[0037] CCK-8 assay for cell proliferation includes: Example 2 CCK-8 assay for ACSL4 overexpression / knockdown AML cell proliferation: Logarithmic growth phase cells were used to prepare single-cell suspensions. 100 μl of each cell was seeded in a 96-well plate, with three replicates and a blank control. At 0, 24, 48, 72, and 96 h, 10 μl of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm (reference 630 nm) was measured using a microplate reader. Proliferation curves were plotted. Data are expressed as mean ± standard deviation, and were independently replicated in triplicate and statistically analyzed.

[0038] Analysis results: such as Figure 2 The figure shows the proliferation of AML cells after ACSL4 overexpression. Figure 3 The image shows the proliferation of AML cells after ACSL4 knockdown. The results indicate that ACSL4 overexpression promotes AML cell proliferation, while ACSL4 knockdown inhibits AML cell proliferation.

[0039] Example 3 CCK-8 assay for the proliferation of IGF2BP3-overexpressing AML cells treated with rosiglitazone: Logarithmic growth phase cells were collected and a single-cell suspension was prepared at a concentration of 1×10⁻⁶.5 / mL. 50 μl was inoculated into five 96-well plates, 50 μl per well. Rosiglitazone drug concentrations of 0 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 150 μM were prepared and added to the 96-well plates in sequence according to the concentration gradient, 50 μl per well, with final drug concentrations of 0, 5 μM, 10 μM, 25 μM, 50 μM, and 75 μM. Three replicates and a blank control were included. At 0, 24, 48, 72, and 96 h of drug treatment, 10 μl of CCK-8 solution was added to each well, and the plates were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm (reference 630 nm) was measured using a microplate reader. Proliferation curves were plotted, and data are expressed as mean ± standard deviation. Data were independently replicated three times and statistically analyzed.

[0040] Analysis results: such as Figure 4 The image shows the cell proliferation of AML cells overexpressing IGF2BP3 treated with rosiglitazone. The results show that rosiglitazone inhibits cell proliferation in a concentration- and time-dependent manner.

[0041] Example 4 CCK-8 assay for the proliferation of IGF2BP3-overexpressing AML cells by PRGL493: Logarithmic growth phase cells were collected and a single-cell suspension was prepared at a concentration of 1×10⁻⁶. 5 / mL. 50 μl was inoculated into five 96-well plates, 50 μl per well. PRGL493 drug concentrations of 0 μM and 20 μM were prepared and added sequentially to the 96-well plates according to the concentration gradient, 50 μl per well, with final drug concentrations of 0 and 10 μM. Three replicates and a blank control were included. At 0, 24, 48, 72, and 96 h of drug treatment, 10 μl of CCK-8 solution was added to each well. After incubation at 37°C in the dark for 2 h, the absorbance at 450 nm (reference 630 nm) was measured using a microplate reader. Proliferation curves were plotted. Data are expressed as mean ± standard deviation, and were independently replicated three times and statistically analyzed.

[0042] Analysis results: such as Figure 5 The image shows the cell proliferation of an IGF2BP3-overexpressing AML cell line treated with PRGL493. The results indicate that PRGL493 inhibits cell proliferation in a time-dependent manner.

[0043] Example 5 CCK-8 assay for the proliferation of primary AML cells / healthy control peripheral blood mononuclear cells treated with rosiglitazone: Logarithmic growth phase cells were collected, and a single-cell suspension was prepared at a concentration of 1×10⁻⁶. 7 / mL. 50 μl was inoculated into five 96-well plates, 50 μl per well. Rosiglitazone drug concentrations of 0 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 150 μM were prepared and added to the 96-well plates in sequence according to the concentration gradient, 50 μl per well, with final drug concentrations of 0, 5 μM, 10 μM, 25 μM, 50 μM, and 75 μM. Three replicates and a blank control were included. At 0, 24, 48, 72, and 96 h of drug treatment, 10 μl of CCK-8 solution was added to each well, and the plates were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm (reference 630 nm) was measured using a microplate reader. Proliferation curves were plotted, and data are expressed as mean ± standard deviation. Data were independently replicated three times and statistically analyzed.

[0044] Analysis results: such as Figure 6 The figure shows the cell viability of primary AML cells after treatment with rosiglitazone. Figure 7 The figure shows the cell viability of peripheral blood mononuclear cells from healthy controls after treatment with rosiglitazone. The results indicate that rosiglitazone reduces the cell viability of primary AML cells in a concentration-dependent manner, and that rosiglitazone has no significant effect on the cell viability of peripheral blood mononuclear cells from healthy controls.

[0045] Flow cytometry analysis of apoptosis includes: Example 6 Flow cytometry analysis of apoptosis in ACSL4-overexpressing / knockdown AML cells: Logarithmic growth phase cells were collected from each group, washed with PBS, resuspended, and the concentration was adjusted to 1-5 × 10⁻⁵. 5 Cells / ml. Take 100 μl of cell suspension, add 2 μl of Annexin V-APC and 2 μl of 7-AAD dye, vortex to mix, and incubate at room temperature in the dark for 15 minutes. After adding 400 μl of binding buffer, analyze the APC and 7-AAD signals by flow cytometry within 1 hour. The experiment was repeated 3 times, and the apoptosis rate was calculated and statistically compared using software such as FlowJo.

[0046] Analysis results: such as Figure 8 The figure shows the apoptosis of AML cells after ACSL4 overexpression, as shown in the figure. Figure 9 The results show the apoptosis of AML cells after ACSL4 knockdown. Overexpression of ACSL4 inhibits AML cell apoptosis, while ACSL4 knockdown promotes AML cell apoptosis.

[0047] Example 7 For primary AML cells / healthy control cells: Freshly extracted bone marrow mononuclear cells from newly diagnosed AML patients were collected, and cell suspensions were prepared and seeded into 6-well plates. The corresponding volume of rosiglitazone stock solution was added to each well to achieve final concentrations of 0, 5 μM, 10 μM, 25 μM, 50 μM, and 75 μM. After 48-72 hours of drug treatment, cells were collected, washed with PBS, resuspended, and the concentration adjusted to 1-5 × 10⁻⁵ cells / well. 6 Cells / ml were collected, and 100 μl of cell suspension was added, along with 2 μl of Annexin V-APC and 2 μl of 7-AAD dye. The mixture was vortexed and incubated at room temperature in the dark for 15 minutes. After adding 400 μl of binding buffer, the cells were analyzed by flow cytometry within 1 hour to assess APC and 7-AAD signals. The experiment was repeated three times, and the apoptosis rate was calculated and statistically compared using software such as FlowJo.

[0048] Analysis results: such as Figure 10 The image shows the apoptosis of primary AML cells induced by rosiglitazone. Figure 11 The image shows the effect of rosiglitazone on apoptosis in peripheral blood mononuclear cells of healthy controls. The results indicate that rosiglitazone promotes apoptosis in a concentration-dependent manner, and that rosiglitazone has no significant effect on apoptosis in peripheral blood mononuclear cells of healthy controls.

[0049] Example 8 Flow cytometry analysis of ACSL4 protein expression was performed as follows: Primary AML cells treated with rosiglitazone for 48-72 hours were collected, washed with PBS, and the supernatant was discarded. 300 μl of freshly prepared 1×Fixation / Permeabilization working solution was added to each sample, and the cells were incubated at room temperature in the dark for 1 hour. 1 ml of freshly prepared 1×Permeabilization Buffer was added to each tube, and the cells were centrifuged at 600g for 5 minutes at room temperature, and the supernatant was discarded. 50 μl of Rabbit anti-ACSL4 Ab (proteintech) prepared at a 1:100 ratio with 1×Permeabilization Buffer was added to each tube, and the cells were incubated at 4°C for 1 hour. 1 ml of freshly prepared 1×Permeabilization Buffer was added to each tube, and the cells were centrifuged at 600g for 5 minutes at room temperature, and the supernatant was discarded. 100 μl of anti-Rabbit Alexa Fluor 647 prepared at a 1:1000 ratio with 1×Permeabilization Buffer was added to each tube, and the cells were incubated at 4°C for 30 minutes. Add 1 ml of freshly prepared 1×Permeabilization Buffer to each tube, centrifuge at 600g for 5 minutes at room temperature, and discard the supernatant. Add 200 μL of 1×FACS buffer to each tube, filter the cells through a 200-mesh nylon membrane, and analyze on the flow cytometer (using the APC channel on an Alexa Fluor 647 BD C6 flow cytometer).

[0050] Analysis results: such as Figure 12 The image shows the flow cytometry analysis of ACSL4 protein expression in primary AML cells after treatment with rosiglitazone. The results indicate that rosiglitazone reduced ACSL4 protein expression in a concentration-dependent manner.

[0051] Example 9 Western blotting analysis of ACSL4 protein expression was performed as follows: Primary AML cells treated with rosiglitazone for 48-72 hours were collected, washed with PBS, and the supernatant was discarded. An appropriate amount of RIPA strong lysis buffer (Beyotime) was added according to the cell volume ratio, along with 1× final concentrations of phosphatase inhibitor and protease inhibitor, and the mixture was gently pipetted to mix. The cells were then sonicated on ice (7 seconds on, 8 seconds off, total 35 seconds), followed by 15 minutes of standing on ice to ensure complete lysis. The cells were centrifuged at 13000 rpm for 15 minutes at 4°C to separate soluble total protein. The supernatant was carefully transferred to a new EP tube, avoiding aspiration of precipitate. Protein concentration was determined using the Bradford method, and a standard curve was constructed using BSA for quantification. The volume V of the protein sample was measured. 样品 According to formula V buffer =V 样品 / 4 Calculate the volume of 5× protein buffer to be added for each sample; add 5× protein buffer and mix well (final concentration 1×); after denaturing at 99℃ for 10 minutes, calculate the loading volume according to the required loading amount and perform SDS-PAGE electrophoresis.

[0052] Analysis results: such as Figure 13 Western blot was used to detect ACSL4 protein expression in primary AML cells after treatment with rosiglitazone. The results showed that rosiglitazone reduced ACSL4 protein expression in a concentration-dependent manner.

[0053] Example 10 The intravenous injection animal model was established using 6-week-old female NCG-immune-deficient mice. Each mouse was injected with 1×10⁻⁶ NCG via the tail vein. 6 MOLM13-Ctrl-luc / MOLM13-IGF2BP3-OE-luc cells were used. Starting on day 5 after modeling, mice were divided into groups and administered drugs daily. The groups were as follows: Ctrl+PBS group (PBS); OE+PBS group (PBS); OE+ROSI group (rosiglitazone 50 mg / kg / qd, oral gavage, ig) for 14 days. Clinical scores of mice were assessed daily, including body weight, activity level, appearance, arched back, and paralysis. Tumor burden was assessed periodically using imaging, and overall survival was observed.

[0054] Analysis results: such as Figure 14 The figure shows the total survival time of mice in different drug administration groups; as shown Figure 15 Clinical scores of rosiglitazone-treated IGF2BP3-overexpressing and control AML mice; such as Figure 16 In vivo imaging of mice in different drug administration groups (days 12 and 19 of modeling); such as Figure 17 Quantification results for in vivo mouse imaging.

[0055] The results showed that mice in the Ctrl+PBS group had the longest survival time, while mice in the OE+ROSI group had a longer survival time compared to the OE+PBS group. Furthermore, the OE+PBS group achieved the highest clinical score earliest, while the Ctrl+PBS and OE+ROSI groups achieved high clinical scores relatively later, indicating that the onset of AML in mice treated with OE+ROSI was relatively delayed. At the same time, on day 19, the Ctrl+PBS and OE+ROSI groups had lower tumor burden, while the OE+PBS group had the highest tumor burden.

[0056] Example 11 Six-week-old female NCG-immune-deficient mice were used, and each mouse was injected with 1×10⁻⁶ NCG via the tail vein. 6MOLM13-shNC / MOLM13-shACSL4 cells were used. After modeling, the clinical scores of mice were assessed daily, including body weight, activity level, appearance, arched back, and paralysis, while the overall survival of the mice was observed.

[0057] Analysis results: such as Figure 18 To investigate the changes in survival time of NCG mice after ACSL4 knockdown. The results showed that ACSL4 knockdown prolonged the overall survival of NCG mice (31 days in the shNC group and 37 days in the shACSL4 group).

[0058] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the appended claims.

Claims

1. The use of ACSL4 inhibitors in the preparation of drugs for the treatment of acute myeloid leukemia.

2. The use of the ACSL4 inhibitor according to claim 1 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The ACSL4 inhibitor targets the ACSL4 protein, inhibiting AML cell proliferation and promoting apoptosis.

3. The use of the ACSL4 inhibitor according to claim 1 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The ACSL4 inhibitor is at least one of rosiglitazone and PRGL493.

4. The use of the ACSL4 inhibitor according to claim 1 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The drug also contains pharmaceutically acceptable excipients.

5. The use of the ACSL4 inhibitor according to claim 4 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The pharmaceutically acceptable excipients are diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants.

6. The use of the ACSL4 inhibitor according to claim 1 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The dosage form of the drug is a solution, sustained-release agent, suspension, granule, tablet, capsule, powder, emulsion, syrup or drops.

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