Application of MI-2 compound in preparation of medicine for treating acute myelogenous leukemia

Abstract

The invention relates to application of an MI-2 compound in preparation of a medicine for treating acute myelogenous leukemia, and belongs to the technical field of biological medicine. It is proved for the first time that in AML cells without MALT1 protease activity, the compound MI-2 (MALT1 inhibitor) can inhibit cell proliferation and induce apoptosis through a brand new mechanism independent of MALT1 inhibition, namely specific regulation and control of lipid metabolism of the AML cells, and therefore the compound MI-2 (MALT1 inhibitor) serves as a candidate drug or a brand new treatment strategy for treating acute myelogenous leukemia for the first time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of MI-2 compound in the preparation of drugs for treating acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem / progenitor cells, characterized by the disordered proliferation and differentiation arrest of abnormal primitive cells in the bone marrow. Despite advances in targeted therapy and novel treatments in recent years, the overall prognosis of AML patients, especially those with relapsed / refractory disease, remains severe. Therefore, exploring new therapeutic targets and developing novel treatment strategies is of great clinical significance. In recent years, tumor metabolic reprogramming has been considered one of the core hallmarks of cancer, with abnormal lipid metabolism playing a key role in the occurrence, development, and drug resistance of AML. Studies have shown that AML cells are highly dependent on endogenous fatty acid synthesis to meet the biomembrane construction and energy storage requirements for their rapid proliferation. Key lipid metabolism enzymes, such as fatty acid synthase and stearoyl-CoA desaturase 1, are often significantly overexpressed in AML cells, and their activity is associated with cell survival and poor prognosis. Therefore, targeting lipid metabolism pathways has become a promising but underdeveloped new direction in the treatment of AML.

[0003] On the other hand, the small molecule compound MI-2 is widely recognized as an effective inhibitor of the MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) protease. In existing technologies, MI-2 is specifically designed and applied to treat B-cell lymphomas dependent on MALT1 protease activity. Its accepted classic mechanism of action is to specifically inhibit the cleavage of downstream substrates (such as A20) by covalently binding to the active site of MALT1, thereby interfering with related signaling pathways. However, recent scientific research reveals that the biological functions of MI-2 may be more complex and diverse than initially thought. Recent literature reports that MI-2 can induce ferroptosis in certain cancer cells by directly inhibiting the key regulator of ferroptosis, GPX4, and this effect has been shown to be independent of its ability to inhibit MALT1 protease activity. This discovery provides the first conclusive evidence that MI-2 possesses "off-target" effects independent of its original target (MALT1 protease) and can influence cell fate through other mechanisms. This suggests that the potential applications of MI-2 as a chemical tool and drug candidate may be far from being fully explored.

[0004] Currently, therapeutic approaches that directly target key nodes in lipid metabolism in acute myeloid leukemia (AML) cells remain scarce. Although recent research (on ferroptosis) has broken the absolutist notion that "MI-2 function is solely equivalent to MALT1 inhibition," this new finding has not yet established any connection with the classic lipid synthesis pathways and treatments in AML. More critically, existing technologies completely lack systematic analysis of the MI-2 mechanism of action within the specific disease model of AML. Therefore, there is an urgent need in this field to address these mechanistic blind spots and fill the following gap: after clearly ruling out the possibility of MALT1 protease activity-mediated intervention, to reveal whether and how MI-2 exerts its anti-leukemic effect through novel mechanisms (especially whether it intervenes in the fatal weakness of AML cells—lipid metabolism), thereby laying a solid scientific foundation for its development as a novel treatment strategy for AML. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of compound MI-2 in the preparation of drugs for treating acute myeloid leukemia (AML). This invention demonstrates for the first time that compound MI-2 (a MALT1 inhibitor) can inhibit cell proliferation and induce apoptosis in AML cells lacking MALT1 protease activity through a novel mechanism independent of MALT1 inhibition—specifically regulating lipid metabolism in AML cells. Therefore, this invention is proposed for the first time as a candidate drug or a novel therapeutic strategy for treating AML.

[0006] Compared with existing technologies, the advantages of this invention are that it has fully constructed a research system for a new model of acute myeloid leukemia that is ineffective against MALT1, verified its therapeutic efficacy, excluded old mechanisms of action through experimental evidence of ineffective MLT-985, clarified a new mechanism for regulating lipid metabolism, and explored new uses for the treatment of acute myeloid leukemia, forming a clear logical chain from mechanism identification to therapeutic application.

[0007] This invention is achieved through the following technical solution:

[0008] The purpose of this invention is to provide the application of MI-2 compound in the preparation of drugs for treating acute myeloid leukemia, wherein the structural formula of MI-2 compound is:

[0009] .

[0010] In one embodiment of the present invention, the dosage of the MI-2 compound is 1 μM-5 μM.

[0011] In one embodiment of the present invention, the drug exerts its effect by inhibiting lipid metabolism in acute myeloid leukemia cells; the acute myeloid leukemia cells are acute myeloid leukemia cells that do not express or lack functional MALT1 protease; the inhibition of lipid metabolism in acute myeloid leukemia cells is manifested by downregulating the expression or activity of fatty acid synthase and / or stearoyl-CoA desaturase 1.

[0012] In one embodiment of the present invention, the drug further includes a chemotherapeutic agent and / or a targeted agent for treating acute myeloid leukemia.

[0013] In one embodiment of the present invention, the dosage form of the drug is tablets, capsules, granules, emulsions, dry suspensions, dry extracts, or injections.

[0014] In one embodiment of the invention, the drug further includes a pharmaceutically or pharmacologically acceptable carrier and / or salt.

[0015] In one embodiment of the present invention, the carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, filler, suspending agent, surfactant, and preservative.

[0016] In one embodiment of the invention, the filler is selected from one or more of starch, sucrose, and lactose; the wetting agent includes glycerin; and the surfactant includes hexadecyl alcohol.

[0017] In one embodiment of the present invention, the adhesive is selected from one or more of cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone.

[0018] In one embodiment of the present invention, the disintegrant is selected from one or more of agar, calcium carbonate, and sodium bicarbonate.

[0019] In one embodiment of the invention, the pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

[0020] This invention provides a complete technical solution from molecular basis and cellular mechanism to animal model validation, systematically revealing the novel use and mechanism of action of compound MI-2 in the treatment of acute myeloid leukemia (AML). Detection confirmed that the AML cell lines used (such as U937) lacked markers of MALT1 protease activity (such as A20 protein cleavage), thus clarifying that this study was conducted in a "MALT1-independent" disease model. At the in vitro cellular level, compound MI-2 significantly inhibited AML cell proliferation, arrested the cell cycle, and induced apoptosis. At the in vivo animal model level, in an AML xenograft severely immunodeficient mouse model constructed from U937-luciferase cells, after treatment with MI-2 via tail vein injection, in vivo imaging technology detected a significant reduction in tumor burden (fluorescence signal) compared to the control group. This result directly demonstrates that MI-2 has definite anti-AML activity in vivo. Experiments such as MLT-985 ineffectiveness and unchanged efficacy in MALT1 knockdown cells confirmed that the in vitro and in vivo activities are independent of the MALT1 protein.

[0021] This invention completely eliminates the MALT1-dependent mechanism. Through transcriptome sequencing analysis of acute myeloid leukemia cells, it is the first time that MI-2 treatment has led to a comprehensive downregulation of lipid metabolism-related gene pathways. The downregulation of key lipid metabolism enzymes was verified at the messenger RNA (mRNA) and protein levels.

[0022] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0023] (1) This invention provides the application of MI-2 compound in the preparation of drugs for the treatment of acute myeloid leukemia. This invention provides a novel and potent candidate drug (MI-2) for the treatment of acute myeloid leukemia, which has been validated by in vivo and in vitro models. It reveals the scientific principle by which it exerts its effects by inhibiting lipid metabolism in acute myeloid leukemia cells, laying a solid theoretical and practical foundation for its further clinical development.

[0024] (2) This invention is the first to creatively apply the known MALT1 protease inhibitor MI-2 to the treatment of acute myeloid leukemia. This application is completely independent of its original design target (MALT1), which solves the problem of the narrow application of MI-2 in the prior art (it is only effective in diffuse large B-cell lymphoma), and realizes the significant value expansion of "repurposing old drugs".

[0025] (3) This invention reveals and verifies a novel mechanism of action that is independent of MALT1. Through the use of the highly selective inhibitor MLT-985 (in U937 cells, the cell survival rate was still >90% after treatment with 10 μM MLT-985 for 48 hours, while the survival rate of MI-2 was <10% under the same conditions) and experiments showing that the activity of MI-2 remained unchanged in MALT1 gene knockdown cells (half-maximum inhibitory concentration change <10%), it is irrefutably demonstrated that the anti-acute myeloid leukemia effect of MI-2 is not related to MALT1 protein and its enzyme activity. In addition, no fragment of the classical cleavage substrate protein A20 of MALT1 was observed in acute myeloid leukemia cell lines.

[0026] Transcriptome sequencing analysis revealed that MI-2 significantly downregulated the lipid metabolism pathway gene set in acute myeloid leukemia cells (Gene set enrichment analysis (GSEA) showed an enrichment fraction of -1.683 for this pathway, with a nominal P-value of 0.001). Western blotting analysis showed that this compound significantly downregulated the expression of core lipid synthesis-related proteins in a dose-dependent manner. After treatment with different concentrations for 48 hours, the levels of key proteins, including fatty acid synthase (FASN), acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element-binding protein 1 (SREBP1), and stearoyl-CoA desaturase 1 (SCD1), all showed significant dose-dependent reductions. In particular, at a concentration of 5 μM, the expression levels of these proteins decreased by more than 50% compared to the control group.

[0027] Consistent with this, real-time quantitative polymerase chain reaction (qRT-PCR) results confirmed this inhibitory effect at the transcriptional level. Similarly, after 48 hours of treatment, the expression levels of messenger ribonucleic acid of genes such as fatty acid synthase, acetyl-CoA carboxylase 1, sterol regulatory element-binding protein 1, and stearoyl-CoA desaturase 1 were also significantly downregulated, with reductions exceeding 50%.

[0028] (4) At both the cellular and animal levels, MI-2 has been shown to have potent anti-acute myeloid leukemia activity, providing a solid empirical basis for its clinical translation.

[0029] In vitro efficacy quantification: Clarify the effects of MI-2 on the proliferation inhibition half-maximal inhibitory concentration (IC50) of acute myeloid leukemia cells (e.g., 2.507 μM for U937 cells), apoptosis induction (increase in cleavage-type PARP and decrease in MCL-1), and cell cycle arrest (increase in the proportion of cells in the quiescent phase and the early stage of DNA synthesis).

[0030] In vivo efficacy quantification: In a mouse model of acute myeloid leukemia xenograft with severe immunodeficiency, in vivo imaging technology was used to quantitatively demonstrate that MI-2 treatment significantly reduced tumor burden (fluorescence signal) in mice (e.g., a reduction of about 80%), providing key preclinical efficacy evidence.

[0031] (5) This invention not only provides MI-2 as a candidate drug molecule, but more importantly, it provides a novel strategy for treating acute myeloid leukemia by targeting the "lipid metabolism addiction" of acute myeloid leukemia cells. This provides a possible new option for acute myeloid leukemia patients who are insensitive to or resistant to existing chemotherapy or targeted drugs, and addresses the urgent need in this field for drugs with new mechanisms of action.

[0032] In summary, through systematic scientific research, this invention not only revolutionizes our understanding and reshapes the value of the known compound MI-2, but also provides an innovative treatment candidate for the major disease of acute myeloid leukemia, which has undergone rigorous mechanism verification and in vitro and in vivo efficacy evaluation, demonstrating significant progress and broad clinical application prospects. Attached Figure Description

[0033] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0034] Figure 1 This is a dose-response curve of the in vitro antileukemic activity of compound MI-2 of the present invention;

[0035] Figure 2 This invention relates to the effects of compound MI-2 on apoptosis and cell cycle in leukemia cell lines MV4-11 and U937; wherein, A is a statistical graph of cell apoptosis detected by flow cytometry; B is a statistical graph of the effect of compound MI-2 on cell cycle; and C is a Western blot map of the expression of apoptosis-related proteins.

[0036] Figure 3 This invention relates to the in vivo therapeutic effect of compound MI-2 in an animal model of leukemia; wherein, A is an in vivo bioluminescence imaging image of tumor-bearing mice after different treatments; B is a quantitative statistical graph of the fluorescence intensity of in vivo imaging in Figure A; C is a statistical graph of the proportion of CD45+ cell infiltration in specific organs (liver, bone marrow, spleen) detected by flow cytometry.

[0037] Figure 4 The diagram shows that the effect of the compound MI-2 of this invention on inhibiting acute myeloid leukemia is independent of the inhibition of MALT1 protease activity; where A is a Western blot analysis diagram; B is a statistical diagram of cell proliferation experiments; and C is the verification of the MALT1 protein knockdown efficiency and the effect of MI-2 intervention on the proliferation of knocked-down and non-knocked cells.

[0038] Figure 5 This is a diagram showing the multi-level research results on the mechanism of action of the compound MI-2 against acute myeloid leukemia in this invention; where A is a gene expression clustering heatmap based on transcriptome sequencing data; B is a functional diagram drawn based on gene set enrichment analysis results; and C is a gene set enrichment analysis curve.

[0039] Figure 6 This document validates the transcriptome sequencing data of compound MI-2 of this invention. Specifically, A is a heatmap classifying the expression patterns of lipid metabolism, cell cycle, and MYC-related genes selected from differentially expressed genes in the transcriptome sequencing; B is a statistical graph of messenger RNA expression levels of lipid metabolism-related genes after MI-2 treatment for different time periods, validated by real-time quantitative polymerase chain reaction (qPCR); C is a statistical graph of messenger RNA expression levels of cell cycle-related genes after MI-2 treatment for different time periods, validated by qPCR; and D is a graph of MYC signal axis phases validated by qPCR. The graph shows the expression levels of messenger RNA of key genes after different treatment times with MI-2; E shows the changes in expression levels of key lipid synthesis proteins, c-MYC protein, and mammalian target of rapamycin complex 1 (MAP1) signaling pathway-related proteins in U937 cells after treatment with different concentrations of MI-2, as detected by Western blotting; F shows the changes in expression levels of fatty acid synthase, stearoyl-CoA desaturase 1, c-MYC, and mammalian MAP1 signaling pathway-related proteins in MV4-11 cells after treatment with different concentrations of MI-2, as detected by Western blotting. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0042] The experimental methods involved in this invention are as follows:

[0043] Cell viability assay (CCK-8 assay): Cells were seeded in 96-well plates, treated with different concentrations of the compound for a specified time, and then 10 μL of CCK-8 solution was added to each well. Cells were cultured for 2-4 hours, and the absorbance was measured at 450 nm using a multi-mode microplate reader. Cell viability (%) = [(absorbance of drug wells - absorbance of blank wells) / (absorbance of control wells - absorbance of blank wells)] × 100%. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 9.0 software.

[0044] Western blotting: Cells were lysed with lysis buffer, and total protein was extracted. Separation was achieved by polyacrylamide gel electrophoresis, and the protein was transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with the corresponding primary antibody, followed by incubation at room temperature for 1 hour with horseradish peroxidase-labeled secondary antibody. Imaging was performed on a chemiluminescence imaging system (Tanon 5200) using an enhanced chemiluminescence kit (Thermo Scientific).

[0045] Flow cytometry analysis of cell cycle and apoptosis: For cell cycle analysis, cells were fixed with pre-chilled 70% ethanol, treated with ribonuclease, stained with propidium iodide, and detected using a flow cytometer (BD FACS Celesta). Cell cycle analysis software was also used. For apoptosis analysis, an annexin V-fluorescein isothiocyanate / propidium iodide double staining apoptosis detection kit was used, following the manufacturer's instructions. Real-time quantitative polymerase chain reaction (qPCR): Total ribonucleic acid (RNA) was extracted from cells using the TRIzol method and reverse transcribed to synthesize RNA. Amplification was performed using SYBR Green premixed buffer on a LightCycler 480 qPCR instrument (Roche). GAPDH was used as an internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCt) method.

[0046] Transcriptome sequencing and bioinformatics analysis: The process was outsourced to a professional company (Novogene) using the Illumina NovaSeq 6000 platform. After quality control and alignment, the raw data were analyzed for differentially expressed genes using DESeq2. Gene set enrichment analysis utilized marker gene sets from a molecular signature database.

[0047] Mouse xenograft tumor model and in vivo imaging: A systemic disseminated acute myeloid leukemia model was established by injecting stably luciferase-expressing U937 cells (U937-luciferase) into 6-8 week old female severely immunodeficient mice via tail vein injection. In the treatment group, starting from day 8 after cell implantation, MI-2 (25 mg / kg, a mixture of 5% dimethyl sulfoxide, 40% polyethylene glycol 300, 5% Tween 80, and 50% physiological saline) was injected intraperitoneally every other day. At designated time points, D-luciferin potassium (150 mg / kg) was injected intraperitoneally, and imaging was performed 10 minutes later using a small animal in vivo optical imaging system (PerkinElmer IVISSpectrum). Total photon flux throughout the body was quantitatively analyzed using Living Image software.

[0048] Example 1: In vitro inhibitory effect of MI-2 on the proliferation of various acute myeloid leukemia cell lines

[0049] This embodiment aims to demonstrate the broad-spectrum antiproliferative activity of MI-2 against acute myeloid leukemia cells in vitro.

[0050] Cell lines: Human acute myeloid leukemia cell lines U937, MV4-11, NB4, HL-60, Kasumi-1, and MOLM-16. All cells were cultured on RPMI-1640 medium (Gibco, catalog number 11875093) containing 10% fetal bovine serum (Sigma-Aldrich, catalog number F0193).

[0051] It was placed in an incubator at 37°C and 5% CO2.

[0052] Compound: MI-2 (purchased from MedChemExpress, catalog number HY-12276), prepared as a 20 mM stock solution with dimethyl sulfoxide, aliquoted and stored at -20°C. Dilute to the required concentration with complete culture medium before use.

[0053] Experimental procedure: Cells in the logarithmic growth phase were seeded into 96-well plates, 5000 cells per well. After attachment, fresh culture medium containing gradient concentrations of MI-2 (0, 1 μM, 3 μM, 5 μM, 10 μM) was added, with three replicates for each concentration. The cells were cultured for another 48 hours.

[0054] Detection method: Cell viability was detected using the CCK-8 assay.

[0055] Specific results:

[0056] like Figure 1As shown, MI-2 significantly inhibited the proliferation of all tested acute myeloid leukemia (AML) cell lines in a concentration-dependent manner. The calculated 48-hour half-maximal inhibitory concentrations (IC50) for each cell line were: U937 cells 2.507 μM, MV4-11 cells 1.394 μM, NB4 cells 0.474 μM, HL-60 cells 1.893 μM, and Kasumi-1 cells 0.01672 μM. These results quantitatively demonstrate that MI-2 possesses potent and broad-spectrum in vitro antiproliferative activity against AML cells.

[0057] Example 2: MI-2-induced apoptosis and cell cycle arrest in AML cells

[0058] This embodiment aims to explore the cellular mechanism by which MI-2 inhibits the proliferation of acute myeloid leukemia cells.

[0059] Cells and compounds: U937 and MV4-11 cell lines were used. The MI-2 stock solution and working solution were prepared in the same manner as in Example 1.

[0060] Apoptosis detection: Cells were stored at 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and treated with MI-2 at concentrations of 0 (dimethyl sulfoxide control), 1 μM, 3 μM, and 5 μM for 48 hours. Cells were collected, stained with annexin V-fluorescein isothiocyanate / propidium iodide apoptosis detection kit, and analyzed by flow cytometry.

[0061] Cell cycle detection: The treatment method is the same as for apoptosis detection. After treatment, cells are collected, stained with propidium iodide, and their DNA content is detected by flow cytometry.

[0062] Detection of related protein expression: After treating cells with the same concentration of MI-2 for 48 hours, total protein was extracted from the cells and analyzed by Western blot. The primary antibodies used included: PARP (catalog number 9542S, purchased from CST), cleavage-type Caspase-3 (catalog number 9661S, purchased from CST), MCL-1 (catalog number 4572S, purchased from CST), and GAPDH (catalog number MAB373, purchased from Millipore).

[0063] Specific results:

[0064] Induction of apoptosis: Flow cytometry results showed that after treatment with 5 μM MI-2 for 48 hours, the apoptosis rates of U937 and MV4-11 cells were significantly higher than those of the control group (p < 0.001). Figure 2 A in the text). Results of Western blotting ( Figure 2(C) shows that with increasing MI-2 concentration, the band intensity of pro-apoptotic proteins cleaved by PARP and cleaved by Caspase-3 in U937 and MV4-11 cells was significantly enhanced, while the band intensity of the anti-apoptotic protein MCL-1 was weakened, confirming the MI-2-induced apoptosis effect at the protein level. Figure 2 (C in the middle).

[0065] Cell cycle arrest: Flow cytometry analysis showed ( Figure 2 After treatment with 3 μM MI-2 for 48 hours, the proportion of U937 and MV4-11 cells arrested in the quiescent phase and the early stage of DNA synthesis significantly increased (U937: from ~45% to ~65%; MV4-11: from ~50% to ~75%), while the proportion of cells in the DNA synthesis phase, late DNA synthesis phase, and mitotic phase decreased accordingly. This indicates that MI-2 inhibits cell proliferation by arresting cell cycle progression.

[0066] Example 3: In vivo animal model to verify the anti-AML efficacy of MI-2

[0067] This embodiment aims to provide direct therapeutic evidence for MI-2 at the live animal level.

[0068] Animal model establishment: 6-8 week old female severely immunodeficient mice were used. 5 × 10⁵ mice were injected via tail vein. 5 A systemic disseminated acute myeloid leukemia model was established using U937-luciferase cells.

[0069] Grouping and Administration: On day 8 after cell implantation, mice were randomly divided into two groups (n=5): a solvent control group and a MI-2 treatment group. The treatment group received intraperitoneal injections of MI-2 (25 mg / kg) every other day for a total of 8 doses. The control group received an equal volume of solvent.

[0070] Efficacy assessment: Tumor fluorescence signals throughout the mice were monitored using an in vivo imaging system before treatment (day 8), during treatment (day 16), and at the end of treatment (day 24), and the total photon flux was quantitatively analyzed.

[0071] Specific results:

[0072] Live imaging ( Figure 3 A) Visually, by day 24, strong bioluminescent signals appeared throughout the body of mice in the control group (especially in the bone marrow, spleen, and liver), while the signals in the MI-2 treatment group mice were significantly weakened.

[0073] Quantitative analysis ( Figure 3Figure B) shows that from day 16, the mean total photon flux in the MI-2 treatment group was significantly lower than that in the control group (p < 0.05). By day 24, the endpoint of the experiment, the whole-body tumor fluorescence signal intensity in the treatment group was reduced by an average of approximately 80% compared to the control group (p < 0.01). These results, with objective and quantifiable data, irrefutably demonstrate that MI-2 can effectively inhibit the progression of acute myeloid leukemia in vivo and has clear therapeutic potential.

[0074] Further flow cytometry analysis ( Figure 3 The results (C) showed that after treatment, the proportion of human CD45+ (hCD45+) acute myeloid leukemia cells in the bone marrow, spleen, and liver of MI-2 group mice was significantly lower than that in the control group, which confirmed the results of in vivo imaging at the cellular level.

[0075] Example 4: Confirmation that the anti-acute myeloid leukemia effect of MI-2 is independent of its classical target MALT1

[0076] This embodiment aims to systematically rule out the possibility that MI-2 exerts its anti-acute myeloid leukemia effect by inhibiting MALT1 protease activity through a complete and rigorous control experimental system.

[0077] Model and control cell selection:

[0078] Experimental model: Acute myeloid leukemia cell lines that do not possess the functional MALT1 protease were selected.

[0079] Positive control: Jurkat cells (a T-lymphocytic leukemia cell line) with known intact MALT1 protease function were selected.

[0080] MALT1 protease activity verification experiment:

[0081] Stimulation conditions: U937 and Jurkat cells were co-stimulated with phorbol ester (PMA, 50 ng / mL) and ionomycin (1 μM) for 2 hours to activate the nuclear factor-κB signaling pathway and induce MALT1 protease activation.

[0082] Detection method: After stimulation, cells were collected, and total protein was extracted for Western blot analysis.

[0083] Antibody and detection target: The presence or absence of the full-length A20 protein (~90 kDaltons) and the characteristic N-terminal fragment (~70 kDaltons) generated after cleavage by activated MALT1 was detected using an antibody that specifically recognizes the N-terminus of the A20 protein (purchased from CST, catalog number D13H3).

[0084] Pharmacological controlled trials of highly selective MALT1 inhibitors:

[0085] Negative control compound: MLT-985 (purchased from MedChemExpress, catalog number HY-142648), a highly selective and irreversible MALT1 protease inhibitor that has been widely documented in the literature.

[0086] Experimental methods: The cytotoxicity of MLT-985 was detected in parallel using the CCK-8 assay in U937 and MV4-11 cells.

[0087] Treatment conditions: Cells were treated with gradient concentrations (0, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM) for 48 hours.

[0088] Genetic validation experiment (MALT1 knockout model):

[0089] Cell construction: Using lentivirus-mediated small hairpin RNA technology, a stable MALT1 knockdown U937 cell line was constructed (MALT1 group knockdown), and cells infected with disordered small hairpin RNA were set up as controls (control group knockdown).

[0090] Knockdown efficiency verification: The effective knockdown of MALT1 at messenger ribonucleic acid and protein levels was confirmed by Western blotting (using MALT1 antibody, purchased from Santa Cruz, catalog number sc-515751) and real-time quantitative reverse transcription polymerase chain reaction.

[0091] Efficacy test: Cell viability was detected by CCK-8 assay after 48 hours of MI-2 treatment in the control group and MALT1 group knockdown cells, and the half-maximal inhibitory concentration was calculated.

[0092] MALT1 protease activity verification results:

[0093] In positive control Jurkat cells stimulated with phorbol ester / iomycin, Western blotting clearly detected the ~70 kDa N-terminal fragment of the A20 protein cleaved from its amino-terminal region, confirming the presence of functional MALT1 protease activity in this system. Under the same stimulation conditions, only the full-length A20 protein was detected in other acute myeloid leukemia cells, with no cleaved fragments observed. Figure 4 (A) This result, combined with MALT1 expression detection, demonstrates that the MALT1 substrate protein A20 is not cleaved in various acute myeloid leukemia cell lines, indicating that these cells lack spontaneous MALT1 protease activity; thus confirming that the core research model of this invention—the acute myeloid leukemia cell line—is a "MALT1-independent" acute myeloid leukemia model lacking functional MALT1 protease. Pharmacological control experiment results:

[0094] In U937 cells, even after treatment with up to 10 μM MLT-985 for 48 hours, cell viability remained above 90%, indicating that specific inhibition of MALT1 had almost no effect on the survival of acute myeloid leukemia cells in this model. Figure 4 (B in the original text). Under identical experimental conditions, MI-2 exhibited strong cytotoxicity even at low micromolar concentrations, with a half-maximal inhibitory concentration (IC50) of approximately 2.5 μM, and cell viability was below 10% at a concentration of 5 μM. Figure 1 ).

[0095] Conclusion: The anti-acute myeloid leukemia activity of MI-2 is far superior to that of MLT-985, directly proving that its effect is not achieved by inhibiting the MALT1 protease.

[0096] Genetic verification results:

[0097] In U937 cells where MALT1 was successfully knocked down (MALT1 group knockdown), the half-maximal inhibitory concentration (IC50) of MI-2 was less than 10% different from that in control cells (control group knockdown). Figure 4 The C in the figure indicates that the inhibitory effect of compound MI-2 on acute myeloid leukemia cells was not significantly changed after the MALT1 gene was knocked out.

[0098] Conclusion: The absence of MALT1 protein did not affect the efficacy of MI-2, further confirming from a genetic perspective that its mechanism of action does not depend on the presence of MALT1 protein.

[0099] Overall conclusion:

[0100] This embodiment first clarifies that the research model (U937) itself does not possess the classical target activity of MI-2. Using highly selective drug-pharmaceutical tools, it was demonstrated that even complete inhibition of this classical target could not reproduce the MI-2 phenotype. Direct removal of the target protein did not alter MI-2 activity. These three interconnected and irrefutable experimental steps together constitute a complete logical system for excluding MALT1 dependence, laying a solid scientific foundation for the subsequent discovery of novel mechanisms.

[0101] Example 5: Revealing a novel mechanism by which MI-2 regulates c-MYC and lipid metabolism through transcriptomics and molecular validation.

[0102] This embodiment aims to systematically elucidate the precise molecular mechanism by which MI-2 functions in acute myeloid leukemia cells through global transcriptomics analysis and multi-level molecular verification, demonstrating that it exerts its anti-leukemic effect by downregulating c-MYC and inhibiting its downstream mammalian target of rapamycin complex 1 signaling and lipid synthesis pathway.

[0103] Global analysis of transcriptome sequencing:

[0104] Treatment conditions: U937 cells were treated with 3 μM MI-2 or an equal volume of dimethyl sulfoxide for 48 hours, with 3 independent biological replicates per group.

[0105] Sequencing and Analysis: Total ribonucleic acid was extracted from collected cells and sequenced by Beijing Novogene Technology Co., Ltd. After quality control, the raw data were aligned to the human reference genome (GRCh38) using HISAT2 software, and gene counting was performed. Differentially expressed gene analysis was conducted using DESeq2 software (threshold: absolute logarithm of fold change > 0.58 and corrected p-value < 0.05).

[0106] Pathway enrichment analysis: Gene set enrichment analysis was performed on differentially expressed genes using the marker gene set of the Molecular Characteristic Database (version 7.5.1) to identify global biological pathways regulated by MI-2.

[0107] Key gene expression validation (messenger RNA level):

[0108] Cells and treatment: U937 and MV4-11 cell lines were used and treated with 0 and 3 μM MI-2 for 24 hours or 48 hours, respectively.

[0109] Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) assay: Total ribonucleic acid (RNA) was extracted using the TRIzol method and reverse transcribed into complementary deoxyribonucleic acid (DNA). Amplification was performed using SYBR Green premixed buffer on a Roche LightCycler 480 RTD system (purchased from Roche). GAPDH was used as an internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCt) method. Genes detected included:

[0110] Lipid metabolism genes: fatty acid synthase, acetyl-CoA carboxylase 1, sterol regulatory element binding transcription factor 1 (encoding sterol regulatory element binding protein 1), sterol regulatory element binding transcription factor 2, and stearoyl-CoA desaturase.

[0111] MYC signaling genes: MYC itself and its classic target genes ornithine decarboxylase 1 and phosphoglycerate kinase 1.

[0112] Primers: All real-time quantitative polymerase chain reaction primer sequences were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0113] Key protein expression and signaling pathway validation (protein level):

[0114] Treatment and sampling: U937 and MV4-11 cells were treated with 0, 1 μM, 2 μM, 3 μM, and 5 μM MI-2 for 48 hours.

[0115] Western blot assay: Cells were collected, and total protein was extracted using RIPA lysis buffer. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a membrane, the cells were incubated overnight at 4°C with the following primary antibody:

[0116] Key proteins in lipid synthesis: fatty acid synthase (FASN, catalog number 10624-2-AP, purchased from Wuhan Sanying Biotechnology Co., Ltd.), acetyl-CoA carboxylase 1 (ACC1, catalog number 3676S, purchased from CST), sterol regulatory element binding protein 1 (SREBP1, which recognizes the precursor p130 and the mature m.SREBP1, catalog number ab3259, purchased from Abcam), and stearoyl-CoA desaturase 1 (SCD1, catalog number 28678-1-AP, purchased from Wuhan Sanying Biotechnology Co., Ltd.).

[0117] Transcription regulator: c-MYC (catalog number 9402, purchased from CST).

[0118] Signaling pathway proteins: phosphorylated S6 ribosomal protein (p-S6, Ser371, catalog number 9208, purchased from CST Biotechnology), total S6 protein (catalog number 9202, purchased from CST Biotechnology), phosphorylated AKT (p-AKT, Ser473, catalog number 28731-1-AP, purchased from Wuhan Sanying Biotechnology Co., Ltd.), and total AKT protein (catalog number 60203-2-Ig, purchased from Wuhan Sanying Biotechnology Co., Ltd.).

[0119] Internal reference: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, catalog number ab128915, purchased from Abcam).

[0120] The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse immunoglobulin G, which was visualized using a Tanon 5200 chemiluminescence imaging system.

[0121] Specific results:

[0122] 1. Global analysis of transcriptome sequencing reveals that MI-2 synergistically inhibits lipid metabolism, cell cycle, and MYC-related pathways. Figure 5 (A, B, C)

[0123] Transcriptome sequencing and gene set enrichment analysis revealed that MI-2 treatment caused a global change in the gene expression profile of U937 cells.

[0124] The most significantly downregulated gene cluster (cluster A) is enriched in core growth and metabolic pathways such as fatty acid metabolism, MYC proto-oncogene target set V1 / version V2, G2 / M phase checkpoints, and mammalian target of rapamycin complex 1 signaling pathway. Figure 5(B in the original text). Gene set enrichment analysis curves further confirmed that the fatty acid metabolism pathway was significantly inhibited (…). Figure 5 The C-value in the study (normalized enrichment score = -1.683, nominal P-value = 0.001) represents the first unbiased omics finding that MI-2 can synergistically interfere with lipid metabolism, MYC signaling, and cell cycle progression in acute myeloid leukemia cells.

[0125] 2. Visualization of functional classification heatmaps of differentially expressed genes from transcriptome sequencing ( Figure 6 A)

[0126] To visually demonstrate the expression patterns of key genes in the aforementioned pathways, significantly altered lipid metabolism, cell cycle, and MYC-related genes were selected from differentially expressed genes identified by transcriptome sequencing, and a classification heatmap was created. Figure 6 (A) in the figure. The figure clearly shows:

[0127] Lipid metabolism genes (such as fatty acid synthase, acetyl-CoA carboxylase 1, sterol regulatory element binding transcription factor 1, and stearoyl-CoA desaturase) are generally downregulated.

[0128] Cell cycle genes (such as cyclin 20, cyclin-dependent kinase 1, and cyclin-dependent kinase 2): are generally downregulated.

[0129] MYC and its target genes (MYC, ornithine decarboxylase 1, phosphoglycerate kinase 1): synchronously downregulated.

[0130] Figure 6 A in Figure 5 The extended visualization of AC in the system demonstrates the synergistic transcriptional repression of different functional gene modules by MI-2.

[0131] 3. Independent real-time quantitative reverse transcription polymerase chain reaction experiments validated transcriptome sequencing findings and revealed timeliness. Figure 6 To independently verify the reliability of transcriptome sequencing and explore kinetics, B, C, and D in the experiment were designed as targeted real-time quantitative reverse transcription polymerase chain reaction experiments.

[0132] Figure B (Verification of lipid metabolism genes): In U937 cells, MI-2 treatment for 24 hours significantly downregulated the messenger RNA levels of genes such as fatty acid synthase, acetyl-CoA carboxylase 1, sterol regulatory element binding transcription factor 1, and stearoyl-CoA desaturase, with a stronger inhibitory effect after 48 hours. This result independently confirms the finding of lipid metabolism pathway inhibition in transcriptome sequencing.

[0133] Figure C (Cell cycle gene verification): Similarly, MI-2 treatment downregulated the messenger RNA expression of cell cycle genes such as cyclin 20, cyclin-dependent kinase 1, and cyclin-dependent kinase 2 in a time- and dose-dependent manner, independently demonstrating transcriptional repression of cell cycle progression.

[0134] Figure D (MYC signaling axis verification): Real-time quantitative reverse transcription polymerase chain reaction clearly showed that the transcription of the MYC gene itself and the expression of its downstream target genes ornithine decarboxylase 1 and phosphoglycerate kinase 1 decreased synchronously and significantly after MI-2 treatment. This provides independent molecular biological evidence for "MI-2 inhibiting the MYC signaling axis".

[0135] Figures B, C, and D, as independent validation experiments, not only confirmed the accuracy of the transcriptome sequencing data, but more importantly, provided the precise time-dependent relationship of gene expression changes, demonstrating the rapid and synergistic nature of the inhibitory effect.

[0136] 4. Confirm the mechanism at the protein level and demonstrate its universality across cell lines ( Figure 6 (E, F)

[0137] To confirm the final functional output of the above transcriptional changes and verify their universality, protein-level analysis was performed in this embodiment.

[0138] U937 cells (E): Western blot analysis showed that MI-2 significantly reduced the levels of key lipid synthesis proteins (fatty acid synthase, acetyl-CoA carboxylase 1, mature sterol regulatory element-binding protein 1, and stearoyl-CoA desaturase 1) and c-MYC protein in a dose-dependent manner, and inhibited the activity of the mammalian target of rapamycin complex 1 signaling pathway (reduction of phosphorylated ribosomal protein S6 and phosphorylated protein kinase B). This completes the closed loop from "omics discovery" to "transcriptional verification" and then to "protein function verification" in U937 cells.

[0139] MV4-11 cells (F): In another acute myeloid leukemia cell line, MI-2 cells dose-dependently downregulated fatty acid synthase, stearoyl-CoA desaturase 1, c-MYC protein, and inhibited mammalian target of rapamycin complex 1 signaling. These results demonstrate that this mechanism is universal in acute myeloid leukemia cells with different genetic backgrounds.

[0140] Overall conclusion:

[0141] This embodiment constructs a complete evidence system with multiple levels and perspectives: 1) Unbiased omics discovery of core pathways ( Figure 5 AC in the middle); 2) Visualization of omics data ( Figure 6(A) 3) Independent targeted experiments validate omics discoveries and reveal dynamics ( Figure 6 BD in the middle); 4) Protein function and universality confirmation ( Figure 6 These data collectively and conclusively demonstrate that MI-2 exerts its anti-acute myeloid leukemia effect by inhibiting mammalian target of rapamycin complex 1 signaling and downregulating c-MYC, thereby leading to a broad inhibition of its downstream master regulatory network of lipid metabolism. The elucidation of this mechanism provides a solid molecular basis for MI-2 as a novel therapeutic strategy for acute myeloid leukemia.

[0142] Comparative Example 1

[0143] To demonstrate that the effect of the compound MI-2 of this invention does not stem from its known MALT1 inhibitory activity, parallel experiments were conducted using the highly selective MALT1 protease inhibitor MLT-985 in this comparative example.

[0144] In U937 cells, MLT-985 was added at the same concentration gradient as MI-2 (0, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM). After treatment for 48 hours, cell viability was detected by CCK-8 assay.

[0145] Specific results: such as Figure 4 The data show that even with treatment with up to 10 μM MLT-985, the survival rate of U937 cells remained above 90%, and no significant cytotoxicity was observed. This contrasts sharply with MI-2, which exhibits potent inhibitory effects at around 2.5 μM.

[0146] Conclusion: This comparative example directly demonstrates that the traditionally considered target of MI-2 (MALT1 protease) has no pharmacological value in the acute myeloid leukemia disease model targeted in this invention. Therefore, the remarkable activity of MI-2 in acute myeloid leukemia must be attributed to a novel mechanism of action independent of MALT1, highlighting the non-obviousness and technological advancement of this invention.

[0147] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of compound MI-2 in the preparation of drugs for treating acute myeloid leukemia, characterized in that, The structural formula of the MI-2 compound is: 。 2. The application according to claim 1, characterized in that, The dosage of the MI-2 compound is 1 μM-5 μM.

3. The application according to claim 1, characterized in that, The drug exerts its effect by inhibiting lipid metabolism in acute myeloid leukemia cells; the acute myeloid leukemia cells are acute myeloid leukemia cells that do not express or lack functional MALT1 protease; the inhibition of lipid metabolism in acute myeloid leukemia cells is manifested by downregulating the expression or activity of fatty acid synthase and / or stearoyl-CoA desaturase 1.

4. The application according to claim 1, characterized in that, The drugs also include chemotherapy drugs and / or targeted drugs for treating acute myeloid leukemia.

5. The application according to claim 1, characterized in that, The dosage form of the drug is tablets, capsules, granules, emulsions, dry suspensions, dry extracts, or injections.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically or pharmacologically acceptable carriers and / or salts.

7. The application according to claim 6, characterized in that, The carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, filler, suspending agent, surfactant, and preservative.

8. The application according to claim 7, characterized in that, The filler is selected from one or more of starch, sucrose, and lactose; the wetting agent includes glycerin; and the surfactant includes hexadecyl alcohol.

9. The application according to claim 7, characterized in that, The adhesive is selected from one or more of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

10. The application according to claim 6, characterized in that, The pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

Images