Application of reagent for down-regulating FTH1 expression and / or inducing ferroptosis in treatment of multiple myeloma

Abstract

The invention provides application of a reagent for down-regulating FTH1 expression and / or inducing ferroptosis in treatment of multiple myeloma, and belongs to the technical field of biological medicine. According to the application disclosed by the invention, the FTH1 which can induce ferroptosis through a c-Maf / USP7 / FTH1 signal channel so as to inhibit growth of MM in vitro and in vivo and play a key role in iron metabolism is proved for the first time, the medicine can significantly down-regulate expression of the FTH1 in a dose-dependent manner, and overexpression of the FTH1 can significantly reverse the inhibition effect of LEN on the activity of MM cells and LEN-induced ferroptosis. In conclusion, the invention provides a brand new path for treating the MM: the c-Maf / USP7 / FTH1 signal path induces ferroptosis, and a new target spot is provided for treatment of the MM.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of an agent that downregulates FTH1 expression and / or induces ferroptosis in the treatment of multiple myeloma. Background Technology

[0002] Multiple myeloma (MM) is a typically incurable plasma cell cancer of the bone marrow. Its occurrence and development are highly dependent on cytokines and growth factors secreted by the bone marrow microenvironment, such as IL-6, insulin-like growth factor I (IGF-1), and B-cell activating factor (BAFF), ultimately promoting myeloma cell growth and resistance to chemotherapy drugs. In MM, malignant cells are plasma cells that secrete monoclonal immunoglobulins, a hallmark of the disease. MM accounts for approximately 10% of hematologic malignancies and is a highly heterogeneous, genetically complex disease with a rising global incidence, leading to relapse and refractory outcomes after treatment. Currently, MM treatment has shifted from traditional cytotoxic chemotherapy to precision-targeted immunotherapy, represented by monoclonal antibodies and chimeric antigen receptor T-cell therapy. These methods not only directly kill tumor cells but also modulate the bone marrow microenvironment upon which they depend for survival, ultimately benefiting MM patients. It is noteworthy that despite some breakthroughs in clinical treatment, MM patients often face the serious problems of tumor recurrence and low survival rates.

[0003] Currently, lenalidomide (LEN) is widely recognized as a first-line clinical treatment for multiple myeloma (MM), demonstrating significant therapeutic efficacy. Mechanistically, LEN is a derivative of thalidomide and a ligand for the E3 ubiquitin ligase cereblon (CRBN) and recombinant CRBN-DNA damage-binding protein 1 (DDB1). It induces the selective ubiquitination and degradation of two lymphoid transcription factors (IKZF1 and IKZF3) through the CRBN–DDB1–CUL4A–ROC1 E3 ubiquitin ligase complex (CRBN–CRL4). IKZF1 and IKZF3 are crucial for MM cell survival, indicating that drug-mediated downregulation of these transcription factors is an important strategy for disease treatment. Furthermore, LEN can effectively promote the improvement of the MM tumor microenvironment by stimulating T cell and NK cell activity, inhibiting cytokine production in stromal cells, reducing the expression of cell surface adhesion molecules, and inhibiting angiogenesis, ultimately achieving the goal of inhibiting MM. Despite its remarkable clinical efficacy, LEN administration is often accompanied by neurotoxicity and drug resistance during the treatment of multiple myeloma (MM). For example, one study showed that LEN treatment was associated with the severity of neuropathy, revealing that up to 50% of myeloma patients receiving long-term LEN treatment developed sensory axonal neuropathy. Furthermore, research has shown that LEN resistance is related to the activation of the WNT / β-catenin signaling pathway, which inhibits apoptosis and necrosis, further demonstrating that targeting MUCI-C is an effective strategy against LEN-resistant MM cells. These studies highlight the potential of LEN in treating MM; however, current research on the pathways and mechanisms of LEN in treating MM may be insufficient, hindering subsequent drug development. Summary of the Invention

[0004] This invention provides the use of an agent that downregulates FTH1 expression and / or induces ferroptosis in the treatment of multiple myeloma. It can induce ferroptosis through the c-Maf / USP7 / FTH1 signaling pathway to treat MM, providing a new target for the treatment of MM.

[0005] This invention provides the application of a reagent that downregulates FTH1 expression in the preparation of drugs for treating multiple myeloma.

[0006] In a preferred embodiment of the present invention, the reagent induces ferroptosis by adjusting the c-Maf / USP7 / FTH1 axis.

[0007] In a preferred embodiment of the present invention, the reagent has at least one of the following functions: binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

[0008] In a preferred embodiment of the present invention, the reagent comprises lenalidomide.

[0009] The present invention also provides the application of an ferroptosis-inducing agent in the preparation of a drug for treating multiple myeloma.

[0010] In a preferred embodiment of the present invention, the iron death includes iron death induced by the c-Maf / USP7 / FTH1 axis.

[0011] In a preferred embodiment of the present invention, the reagent has at least one of the following functions: binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

[0012] In a preferred embodiment of the present invention, the reagent comprises lenalidomide.

[0013] The present invention also provides a drug for treating multiple myeloma, wherein the active ingredient of the drug has at least the following effects: inducing ferroptosis, binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

[0014] In a preferred embodiment of the present invention, the active ingredient of the drug includes lenalidomide.

[0015] Beneficial Effects: This invention demonstrates for the first time that ferroptosis can be induced through the c-Maf / USP7 / FTH1 signaling pathway, thereby inhibiting MM cell growth in vitro and in vivo. Experiments using LEN as an example showed that LEN directly binds to c-Maf, subsequently inhibiting USP7 transcription levels, thereby inducing FTH1 degradation in MM cells via the ubiquitin-proteasome pathway. Furthermore, in one embodiment, the ferroptosis inhibitor Fer-1 was found to significantly reverse the inhibitory effect of LEN on MM cell viability, indicating that LEN inhibits MM cell growth by inducing ferroptosis. Specifically, LEN can induce the accumulation of ROS and MDA, GSH depletion, decreased mitochondrial membrane potential, and iron overload in MM cells.

[0016] FTH1, which plays a crucial role in iron metabolism, can be significantly downregulated by LEN in a dose-dependent manner. Overexpression of FTH1 can significantly reverse the inhibitory effect of LEN on MM cell viability and LEN-induced ferroptosis. In summary, this invention provides a novel pathway for the treatment of MM: the c-Maf / USP7 / FTH1 signaling pathway induces ferroptosis, providing a new target for the treatment of MM. Attached Figure Description

[0017] Figure 1 Figure showing the results of CCK8 assay for the activity of LEN in H929 cells treated with CCK8. Figure 2 Figure showing the results of LEN-treated MM.1S cells measured by CCK8 assay; Figure 3 Figure showing the results of flow cytometry analysis of apoptosis levels in LEN-treated H929 cells; Figure 4 Figure showing the results of flow cytometry analysis of apoptosis levels in LEN-treated MM.1S cells; Figure 5 Figure showing the LDH levels in the supernatant of MM cells treated with different concentrations of LEN; Figure 6 Figure showing the results of flow cytometry analysis of the activity levels of H929 cells treated with different concentrations of LEN; Figure 7 Image showing the results of mitochondrial morphological changes in LEN-treated H929 cells as detected by transmission electron microscopy. Figure 8 Image showing the results of mitochondrial morphological changes in LEN-treated MM.1S cells as detected by transmission electron microscopy. Figure 9 Figure showing the results of flow cytometry analysis of Lipid ROS levels in LEN-treated H929 cells; Figure 10 Figure showing the results of flow cytometry analysis of Lipid ROS levels in LEN-treated MM.1S cells; Figure 11 Figure showing the results of CCK8 assay of the activity of LEN combined with different cell death inhibitors in H929 cells; Figure 12 Figure showing the results of CCK8 assay of the activity of LEN combined with different cell death inhibitors in H929 cells; Figure 13 To detect MDA and Fe in H929 cells treated with different concentrations of LEN using ELISA. 2+ GSH level results chart; Figure 14 Figure showing the results of mitochondrial membrane potential levels in LEN-treated H929 cells detected by the JC-1 method; Figure 15 Figure showing the results of Western blotting (WB) detection of protein levels of ferroptosis-related molecules in H929 cells treated with different concentrations of LEN; Figure 16 Figure showing the results of CCK8 assay of LEN activity against H929 cells after FTH1 overexpression; Figure 17 Figure showing the results of flow cytometry analysis of Lipid ROS levels in H929 cells after LEN treatment following FTH1 overexpression; Figure 18 Figure 1 shows the results of ELISA detection of MDA, Fe, 4-HNE, and GSH levels in H929 cells treated with LEN after FTH1 overexpression. Figure 19Figure showing the results of qPCR detection of FTH1 mRNA levels in H929 cells after treatment with different concentrations of LEN; Figure 20 The results of Western blotting for detecting the half-life of FTH1 in LEN-treated H929 cells; Figure 21 Figure showing the results of Western blotting to detect the decrease in FTH1 protein levels in LEN-treated H929 cells; Figure 22 The result of Western blotting of FTH1 ubiquitination level in H929 cells after LEN treatment; Figure 23 Figure 1 shows the results of Western blotting of the levels of USP7, USP20, OTUD4 and OTUD6B in H929 cells before and after treatment with different concentrations of LEN. Figure 24 Figure showing the results of Western blotting of FTH1 levels in H929 cells after USP20 knockdown; Figure 25 Figure showing the results of Western blotting of FTH1 levels in H929 cells after OTUD4 knockdown; Figure 26 Figure showing the results of Western blotting of FTH1 levels in H929 cells after OTUD6B knockdown; Figure 27 Figure showing the results of Western blotting analysis of FTH1 levels in H929 cells before and after USP7 overexpression; Figure 28 Figure 1: KEGG enrichment analysis results of H929 cells before and after LEN treatment; Figure 29 Figure 1 shows the GO analysis results of H929 cells before and after LEN treatment; Figure 30 A diagram showing the results of predicting potential transcription factor binding sites in the USP7 promoter region for the JASPAR database; Figure 31 Figure showing the results of CUT&RUN detection of c-Maf binding to the promoter region of USP7 in H929 cells; Figure 32 The results of detecting c-Maf-enhanced USP7 promoter activity for luciferase reporter gene assays; Figure 33 The results of molecular docking detection show that LEN and c-Maf can directly bind; Figure 34 The image shows the results of Western blotting to detect the resistance of LEN-treated c-Maf to streptomycin degradation. Figure 35 The image shows the results of SPR detection of the binding of LEN to c-Maf; Figure 36The graph shows the results of CCK8 assays to determine the activity of LEN on H929 cells after c-Maf overexpression; Figure 37 The figure shows the results of ELISA detection of MDA, Fe, 4-HNE, and GSH levels in H929 cells treated with LEN after c-Maf overexpression; Figure 38 Figure showing the results of flow cytometry analysis of Lipid ROS levels in H929 cells treated with LEN after c-Maf overexpression; Figure 39 Photographs of tumors in myeloma mice after different grouping and treatments; Figure 40 The image shows the results of tumor weight measurement in mice with myeloma. Figure 41 The image shows the results of tumor volume detection in mice with myeloma. Figure 42 A graph showing the results of ELISA detection of ferroptosis markers in tumor tissue; Figure 43 Figure showing the results of immunohistochemical detection of the expression levels of Ki67, c-Maf, USP7, and FTH1 in tumor tissues of myeloma mice; Figure 44 The figure shows the results of Western blotting (WB) analysis of the expression levels of c-Maf, USP7, and FTH1 in myeloma mouse tumor tissue. Detailed Implementation

[0018] This invention provides the application of a reagent that downregulates FTH1 expression in the preparation of drugs for treating multiple myeloma.

[0019] The reagent of the present invention induces ferroptosis by regulating the c-Maf / USP7 / FTH1 axis. Specifically, the reagent has at least one of the following functions: binding to c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

[0020] The reagents described in this invention include LEN. Mechanistic studies have revealed that LEN induces ferroptosis through the c-Maf / USP7 / FTH1 signaling pathway, thereby inhibiting the growth of MM in vitro and in vivo.

[0021] The present invention also provides the application of an ferroptosis-inducing agent in the preparation of a drug for treating multiple myeloma.

[0022] The ferroptosis described in this invention includes ferroptosis induced by the c-Maf / USP7 / FTH1 axis, and the reagent has at least one of the following effects: binding to c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression. The reagent described in this invention includes LEN.

[0023] The present invention also provides a drug for treating multiple myeloma, wherein the active ingredient of the drug has at least the following effects: inducing ferroptosis, binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

[0024] The active ingredient of the drug described in this invention includes LEN.

[0025] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the use of an agent that downregulates FTH1 expression and / or induces ferroptosis in the treatment of multiple myeloma, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, all materials used in the embodiments of this invention are conventional commercially available materials in the art. H929 and MM1S cells were purchased from Guangzhou Lige Technology Co., Ltd.; reagents such as ferroptosis inhibitor (Fer-1), apoptosis inhibitor (zVAD), pyroptosis inhibitor (3-MA), necroptosis inhibitor (BAY), and autophagy inhibitor (NSA) were purchased from MCE. The experimental methods used in the embodiments of this invention are as follows: (1) CCK-8 testing: 1×10⁻⁶ per well 4 MM cells (MM1S and H929 cells) were seeded into 96-well plates and incubated with different concentrations (0, 1, 2, 5, 10, 15 µM) of LEN (MCE) for 24 h, 48 h, and 72 h, respectively. After the incubation time, CCK8 (Vazyme) reagent was added, and after 2 h of incubation, the absorbance at 450 nm was measured using a microplate reader to determine cell viability and calculate the IC50. 50 value.

[0027] (2) Apoptosis detection: MM cells (MM1S and H929 cells) were treated with different concentrations (0, 1, 2, 5, 10, 15 µM) of LEN for 48 h, and then the apoptosis level of cells after treatment with different concentrations of LEN was determined by flow cytometry using an apoptosis kit (Vazyme).

[0028] (3) LDH release assay: An appropriate amount of MM cells (MM1S and H929 cells) were seeded into 96-well cell culture plates, ensuring the cell density did not exceed 80-90% at the time of assay. The plates were then incubated with different concentrations of LEN (0µM, 1µM, 2µM, 5µM, 10µM, 15µM) for 48 hours. One hour before the scheduled assay time, the cell culture plates were removed from the incubator, LDH release reagent (Beyotime) was added, and the plates were repeatedly pipetted to mix thoroughly. The plates were then incubated in the incubator. After the scheduled time, the cell culture plates were centrifuged at 400g for 5 minutes. 120 μL of the supernatant from each well was taken and added to the corresponding well of a new 96-well plate, and the samples were then analyzed.

[0029] (4) PI staining detection: MM cells (MM1S and H929 cells) were treated with different concentrations (0, 1, 2, 5, 10, 15 µM) of LEN for 48 h. After the cells were collected, they were incubated with PI dye (MCE) for 15 min. Then, the cell viability was measured by flow cytometry using the PE channel.

[0030] (5) TEM detection: MM cells were divided into a control group (LEN 0) and an experimental group (LEN 5). After drug treatment for 48 h, the cells were collected and fixed in pre-cooled 2.5% glutaraldehyde fixative at 4℃ for 2-4 hours to initially maintain the ultrastructure. Then, dehydration and embedding were performed. Ultrathin sections of 60-80 nm were cut using an ultramicrotome and attached to a copper grid. The sections were stained with uranium acetate saturated solution for 30 minutes and lead citrate for 5-10 minutes (in the dark). Under a transmission electron microscope, the cell regions were first located at low magnification, and then the mitochondria were observed at high magnification (8000-20000 times).

[0031] (6) Detection of ferroptosis levels: Two types of MM cells were cultured at 2 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 6-well plates and incubated with different concentrations (0µM, 1µM, 2µM, 5µM, 10µM, 15µM) of LEN for 48 h. Cell culture supernatants were then collected, and the levels of ferroptosis-related markers, including MDA (Beyotime, S0131S), 4-HNE (Sangon Biotech, D751041-0048), and GSH (Beyotime, S0053), were detected using ELISA. Simultaneously, cells from each group were collected and stained with the DCFH-DA probe (MCE, HY-D0940). Intracellular ROS levels were detected and analyzed using flow cytometry. The levels of intracellular lipid peroxidation were detected and analyzed using the BODIPY 581 / 591 C11 probe (MCE, HY-D1301).

[0032] (7) JC-1 detection in mitochondria: MM cells were stored at 2 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 24-well plates and incubated with different concentrations (0µM, 1µM, 2µM, 5µM, 10µM, 15µM) of LEN for 48 h. Then, JC-1 reagent (Beyotime, C2006) was added to each well and incubated at 37°C for 2 h. Changes in mitochondrial membrane potential were detected by flow cytometry.

[0033] (8) Western blot (WB) detection: MM cells were treated with different concentrations of LEN (0, 1, 2, 5, 10, and 15 μM) for 48 h. Cells were collected after treatment. The collected cells were mixed with protease and phosphatase inhibitors in RIPA buffer (100:1 ratio), centrifuged, and the supernatant was collected for protein extraction. Protein levels under different treatment conditions were then detected by electrophoresis, transfer, blocking, and antibody coating. For tumor tissue samples, approximately 100 mg of tumor tissue was taken, and total protein was extracted and detected according to the above method. The proteins detected mainly included: GPX4, SLC7A11, ACSL4, ACSL3, CD71, FSP1, FTH1, USP7, c-Maf, USP20, OTUD4, and OTUD6B.

[0034] (9) qPCR analysis: Extract total RNA from the sample (using the TRIzol method or a kit) and determine RNA purity. Use RNA as a template to synthesize cDNA using reverse transcriptase. For each well, use 10 μL of 2×SYBR Green Mix, 0.4 μL each of forward and reverse primers (10 μmol / L), 2 μL of cDNA template, and 7.2 μL of enzyme-free water. Set the PCR reaction program. Process the data after the reaction. The main molecular markers to be detected include FTH1 and all deubiquitinating enzymes.

[0035] FTH1-F (SEQ ID No.1): 5'-ATGACCGAGTCTGCTGCTTT-3', FTH-R (SEQ ID No. 2): 5'-TCACTTGTCCACGATGGTCT-3'.

[0036] (10) Cut & Run Detection: H929 cells in logarithmic growth phase were lysed to obtain nuclei and divided into two groups: the experimental group was given anti-c-Maf antibody, and the control group was given IgG. After incubation at 4℃, pA / G-MNase was added, CaCl2 was used to initiate enzyme digestion, and EGTA was used to terminate it. Proteinase K was used to degrade the protein, and DNA was extracted. qPCR was performed using USP7 promoter-specific primers. The amplification efficiency of the experimental group was significantly higher than that of the control group, which proved the binding.

[0037] (11) Luciferase reporter gene detection: A luciferase reporter plasmid containing the promoter sequence of the USP7 gene transcription start site upstream and downstream of -2000bp to +200bp was constructed. Simultaneously, a c-Maf overexpression plasmid was constructed, with the empty plasmid serving as a negative control. The reporter plasmid, c-Maf overexpression plasmid, and Renilla luciferase internal control plasmid were co-transfected into H929 cells. Gradient-dose LEN treatment groups and a solvent control group were established. Cells were lysed 48 h after transfection, and dual-luciferase activity was detected and relative activity values ​​were calculated.

[0038] (12) Molecular docking: To assess the binding energy and interaction mode between LEN and the c-Maf protein, the molecular structure of LEN was obtained from the PubChem compound database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the protein structure of c-Maf was downloaded from the Uniprot database (https: / / www.uniprot.org / ). Pymol 2.3.0 was used to remove the protein's water of crystallization and original ligands. The protein structure was then imported into AutoDocktools (v1.5.6) for hydrogenation, charge calculation, charge assignment, atom type specification, and saved as a "pdbqt" file. Molecular docking analysis was performed using Autodock Vina 1.2.2 (http: / / autodock.scripps.edu / ) to visualize the molecular docking model.

[0039] (13) CETSA assay: H929 cells in logarithmic growth phase were divided into LEN treatment group and DMSO control group for incubation. After the cells were divided into equal parts, they were subjected to gradient heat shock (40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73℃), and the reaction was terminated by ice bath. The cells were lysed and centrifuged to collect the supernatant. The c-Maf protein content in the supernatant was detected by Western blotting. The results showed that the c-Maf residual amount in the LEN treatment group was significantly higher at high temperature, confirming that LEN binds to c-Maf and improves its thermal stability.

[0040] (14) SPR assay showed that the recombinant human c-Maf protein was expressed and purified in prokaryotic / eukaryotic regions. The purity was verified by SDS-PAGE. Gradient concentrations of LEN solutions were prepared, with a blank buffer used as a control. The c-Maf protein was immobilized on the surface of the SPR sensor chip using the amino-coupling method to ensure a stable immobilization amount. Irrelevant proteins were immobilized in the blank channel as a negative control. Different concentrations of LEN solutions were injected into the chip channels sequentially at a constant flow rate. The changes in the resonance signal (RU value) were monitored in real time, and the binding and dissociation phase curves were recorded. After the experiment, residual molecules on the chip surface were eluted with regeneration solution. The affinity constant was calculated by fitting the curves using software.

[0041] (15) Establishment of a mouse model of multiple myeloma: The mouse type required for this experiment was C57BL / KaLwRij, and 5TGM1 cells were selected and cultured at a rate of 1×10⁻⁶ cells per cell line. 6 Subcutaneous tumor grafts were implanted into mice, and after tumor formation, the mice were randomly divided into three groups: a control group, a LEN treatment group, and a LEN+DFO treatment group. Mice bearing tumors were treated with the drug every two days, while the control group received the same volume of physiological saline. Treatment continued until day 28. Mice were euthanized, and tumor tissue was collected from each group for subsequent related indicator detection. Tumor volume detection: Starting from day 10, the tumor volume of each experimental group was measured every two days, and the length and width of the tumor were recorded. The tumor volume was then calculated using the following formula: Tumor volume = length × width × width / 2, and statistical analysis was performed. Ki67 IHC detection in tumor tissue: After the experiment, tumor tissue was collected from each experimental group, and the expression of Ki67 protein in the tumor tissue was detected using immunohistochemistry. Detection of ferroptosis-related indicators in tumor tissue: After the experiment, tumor tissue was collected from each experimental group, ground, centrifuged, and the supernatant was obtained. The relative contents of MDA, 4-HNE, and GSH were detected by ELISA.

[0042] Example 1: LEN exhibits significant cytotoxicity against multiple myeloma cells. To evaluate the inhibitory effect of LEN on MM cells, this invention conducted several in vitro experiments, including CCK-8 assay, apoptosis assay, LDH release assay, and PI staining assay. The results showed that in LEN-treated H929 and MM1S cells, cell viability was significantly reduced in a time- and dose-dependent manner. Figure 1-2 Furthermore, after 24 hours of incubation, LEN showed an IC50 effect on H929 cells. 50 The effective concentration was 5.413 μM for MM1S cells and 6.206 μM for MM1S cells. Therefore, a concentration of 5 μM was chosen for subsequent experiments to explore the mechanism by which LEN inhibits MM cell activity.

[0043] Next, to assess whether LEN inhibits MM cell activity via the apoptosis pathway, flow cytometry was used to detect the apoptosis rate in MM cells. Data showed that the percentage of apoptotic cells did not differ significantly between H929 and MM1S cells treated with different concentrations of LEN. Figure 3-4 LDH release assays showed that LDH levels released from LEN-treated MM cells increased significantly in a dose-dependent manner. Figure 5 Furthermore, PI staining analysis showed that LEN-mediated MM cell death rate increased significantly in a dose-dependent manner, indicating that LEN had a considerable inhibitory effect on MM cells. Figure 6In summary, LEN is an effective inducer of MM cell death in a dose- and time-dependent manner.

[0044] Example 2: LEN-mediated cytotoxicity is closely related to ferroptosis in MM cells. To further explore the effect of LEN on ferroptosis in MM cells, this invention first evaluated the changes in mitochondrial morphology in MM cells treated with and without 5 μM LEN. TEM results showed that after 24 hours of treatment with 5 μM LEN, both H929 and MM1S cells exhibited an increase in individual mitochondrial networks and a decrease in average branch length, indicating that mitochondrial structure was damaged in LEN-treated MM cells. Figure 7-8 Flow cytometry results showed that in LEN-treated MM cells, the accumulation of Lipid ROS increased in a dose-dependent manner. Figure 9-10 ).

[0045] In addition, to confirm that LEN-inhibited cell activity depends on ferroptosis in MM cells, a CCK-8 assay was performed to assess MM cell viability in the presence of LEN and various inhibitors of different cell death processes (Fer-1, zVAD, 3-MA, BAY, or NSA). Figure 11-12 The results showed that, compared with the LEN-treated group, Fer-1 significantly alleviated the inhibitory effect of LEN on cell viability in H929 and MM1S cells, while no similar phenomenon was observed when co-treated with other inhibitors. Furthermore, the Fer-1-mediated recovery of cell viability was comparable to that of the control group (DMSO-treated) MM cells, with no significant difference. Additionally, the results indicated that LEN treatment significantly increased MDA levels in MM cells and enriched intracellular Fe. 2+ Concentration, and reduced GSH levels in a dose-dependent manner ( Figure 13 ).

[0046] To further elucidate the effects of LEN on mitochondria, mitochondrial membrane potential (Δψm) was assessed using JC-1 staining. The results showed that LEN treatment led to an increase in JC-1 monomer levels and a decrease in JC-1 aggregate levels in H929 cells. Figure 14 In summary, this suggests that LEN can trigger ferroptotic cell death in MM cells, providing a new perspective for LEN treatment of MM.

[0047] Example 3: LEN promotes ferroptosis by reducing the stability of FTH1 in MM cells. This invention used Western blotting to explore potential protein targets of LEN in MM cells, including ACSL3, ACSL4, CD71, GPX4, FSP1, CD98, SLC7A11, and FTH1. Figure 15The results showed that LEN significantly downregulated FTH1 expression in a dose-dependent manner, while having no effect on other proteins.

[0048] Table 1 Information on target names

[0049] To investigate whether FTH1 plays a key role in LEN-induced ferroptosis, this invention transiently overexpressed FTH1 in H929 cells, and then used Western blotting to measure the effect of FTH1 overexpression. Subsequently, CCK-8 assay showed that FTH1 overexpression significantly abolished LEN-induced growth inhibition. Figure 16 ).

[0050] To further investigate the effect of FTH1 on LEN-induced ferroptosis in H929 cells, several biochemical characteristics of ferroptosis were examined, including LipidROS, MDA, and Fe. 2+ The generation of 4-HNE and GSH ( Figure 17-18 The results showed that overexpression of FTH1 eliminated the ROS, MDA, and Fe2+ induced by LEN in H929 cells. 2+ The accumulation of 4-HNE and the reduction of GSH were observed. Overall, these results suggest that LEN induces ferroptosis in MM cells by downregulating FTH1.

[0051] qPCR analysis showed no significant change in FTH1 mRNA levels in LEN-treated MM cells. Figure 19 This indicates that LEN can regulate FTH1 at the protein level. Therefore, it is speculated that LEN reduces FTH1 expression through a posttranscriptional mechanism, meaning that LEN may reduce the stability of FTH1 protein in MM cells.

[0052] After treatment with actinomycin (CHX) for a specified time, Western blot analysis showed that the half-life of FTH1 was significantly shortened in cells co-treated with 5 μM LEN, indicating that LEN treatment accelerated CHX-induced FTH1 protein degradation. Figure 20 ).

[0053] To determine whether LEN mediates FTH1 proteolysis via the ubiquitin-proteasome system or the lysosomal proteolytic pathway (autophagy), MM cells were treated with 10 μM MG132, 10 nM BafA1, or 10 μM CQ in the absence or presence of 5 μM LEN. Results showed that the proteasome inhibitor MG132 significantly rescued LEN-mediated FTH1 protein inhibition, while the autophagy inhibitors BafA1 or CQ did not produce similar results. Figure 21Finally, Co-IP analysis using the anti-FTH1 antibody showed that LEN significantly enhanced the ubiquitination of FTH1 protein in a dose-dependent manner. Figure 22 In summary, LEN regulates FTH1 protein levels in MM cells via the proteasome pathway, inducing ferroptosis by accelerating FTH1 ubiquitination and degradation.

[0054] Example 4: LEN induces FTH1 ubiquitination and degradation by reducing the deubiquitinating enzyme USP7. To determine how LEN affects FTH1 stability via the proteasome pathway, this invention focused on the mRNA expression of deubiquitinating enzymes (DUBs) in LEN-treated H929 cells. Data showed that LEN significantly inhibited USP7, USP20, OTUD4, and OTUD6B compared to the control group (DMSO treatment). Subsequently, Western blotting analysis revealed that LEN significantly reduced the protein levels of USP7, USP20, OTUD4, and OTUD6B in H929 cells in a dose-dependent manner. Figure 23 ).

[0055] Table 2 Genetic information for target names

[0056] To further confirm the role of DUBs in LEN-mediated FTH1 downregulation in MM cells, knockdown of USP7, USP20, OTUD4, and OTUD6B was constructed and confirmed in H929 cells. Western blot analysis showed that significant inhibition of FTH1 expression was detected in USP7 knockdown H929 cells, while similar results were not observed in cells deficient in USP20, OTUD4, or OTUD6B. Figure 24-26 ).

[0057] To demonstrate that LEN mediates FTH1 degradation by inhibiting USP7, this invention subsequently investigated whether USP7 overexpression could rescue the inhibitory effect of LEN on FTH1 expression in H929 cells. The results showed that, compared to the control group (CTRL), USP7 overexpression significantly increased FTH1 expression in MM cells treated with the same concentration of LEN, indicating that LEN-inhibited FTH1 expression in MM cells is USP7-dependent. These results demonstrate that LEN inhibits USP7-induced FTH1 ubiquitination and degradation (…). Figure 27 ).

[0058] Example 5: LEN inhibits USP7 expression by blocking c-Maf-mediated USP7 transcription. To explore how LEN inhibits USP7 expression, this invention first used RNA sequencing (RNA-seq) to analyze transcriptomic changes in LEN-treated H929 cells. KEGG pathway enrichment analysis showed that LEN treatment significantly affected several key pathways, including the TNF signaling pathway, NF-κB signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, and Th17 cell differentiation. Figure 28 Furthermore, GO analysis showed that LEN treatment significantly affected multiple biological processes, including cytokine activity, chemokine receptor binding, and chemokine activity and cytokine receptor binding. Figure 29 Heatmap analysis showed that LEN treatment significantly downregulated USP7 mRNA levels. Furthermore, qPCR confirmed that LEN significantly inhibited USP7 mRNA levels in a dose-dependent manner. To determine whether LEN affects USP7 expression through transcriptional regulation, the JASPAR database was used to predict potential transcription factor binding sites in the USP7 promoter region. The results showed that c-Maf is a potential transcription factor in the USP7 promoter region (…). Figure 30 ).

[0059] To verify this hypothesis, this invention performed cut & run tests, and the results showed that c-Maf can directly bind to the promoter region of USP7 in H929 cells. Figure 31 Furthermore, luciferase reporter gene assays showed that c-Maf overexpression significantly enhanced USP7 promoter activity, while LEN treatment significantly inhibited c-Maf-induced USP7 promoter activity in a dose-dependent manner. Figure 32 Furthermore, Western blot (WB) and qPCR analyses showed that LEN significantly suppressed c-Maf protein and mRNA levels in a dose-dependent manner. Overall, these results indicate that LEN inhibits USP7 transcription by suppressing c-Maf expression.

[0060] Example 6: LEN directly binds to c-Maf To explore how LEN inhibits c-Maf expression, this invention first simulated the binding of LEN to c-Maf through molecular docking. Data showed that the binding energy between LEN and c-Maf was -7.5 kcal / mol, indicating a strong interaction between them. Furthermore, binding site analysis revealed that LEN forms hydrogen bonds with residues in c-Maf (including GLN-305, GLU-309, ARG-312, ALA-313, GLY-314, ASN-315, ARG-316, SER-317, LYS-319, LYS-320, GLN-321, and GLN-322). Figure 33Next, CETSA assays were performed to evaluate the binding of LEN to c-Maf. Data showed that LEN treatment significantly improved the thermal stability of c-Maf compared to the DMSO-treated control group. Furthermore, DARTS assays indicated that LEN treatment significantly enhanced the resistance of c-Maf to streptomycin degradation. Figure 34 Finally, SPR analysis showed that LEN binds to c-Maf with an equilibrium dissociation constant (KD) of 1.62 × 10⁻⁶. -6 M ( Figure 35 In summary, these results indicate that LEN binds directly to c-Maf.

[0061] Example 7: LEN induces ferroptosis via the c-Maf / USP7 / FTH1 signaling pathway To further confirm that LEN induces ferroptosis through the c-Maf / USP7 / FTH1 signaling pathway, several in vitro experiments were conducted. First, Western blot analysis showed that overexpression of c-Maf significantly reversed the inhibitory effect of LEN on USP7 and FTH1 expression. Furthermore, CCK-8 assays indicated that overexpression of c-Maf significantly reversed the inhibitory effect of LEN on H929 cell viability. Figure 36 Furthermore, data showed that overexpression of c-Maf significantly reversed the LEN-induced LipidROS, MDA, and Fe2+ levels in H929 cells. 2+ The accumulation of 4-HNE and the reduction of GSH ( Figures 37-38 Finally, Co-IP assays showed that overexpression of c-Maf significantly reversed LEN-induced FTH1 ubiquitination in H929 cells. In summary, these results indicate that LEN induces ferroptosis through the c-Maf / USP7 / FTH1 signaling pathway.

[0062] Example 8: LEN induces ferroptosis in vivo via the c-Maf / USP7 / FTH1 signaling pathway To evaluate the therapeutic effect of LEN on MM in vivo, a mouse xenograft model was established in this invention. Compared with the control group, LEN treatment significantly inhibited tumor growth, while co-treatment with LEN and DFO significantly reversed the inhibitory effect of LEN on tumor growth. Figure 39 Furthermore, compared to the control group, LEN treatment significantly reduced tumor weight, while co-treatment with LEN and DFO significantly reversed the inhibitory effect of LEN on tumor weight. Figure 40 ).

[0063] Furthermore, compared with the control group, LEN treatment significantly reduced tumor volume, while co-treatment with LEN and DFO significantly reversed the inhibitory effect of LEN on tumor volume. Figure 41Furthermore, compared to the control group, LEN treatment significantly increased MDA and Fe in tumor tissue. 2+ The levels of 4-HNE and GSH were reduced, while co-treatment with LEN and DFO significantly reversed the effects of LEN on MDA and Fe. 2 + The regulatory effects of 4-HNE and GSH levels ( Figure 42 Next, IHC analysis showed that, compared with the control group, LEN treatment significantly reduced the expression of c-maf, USP7, and Fth1, while co-treatment with LEN and DFO significantly reversed the inhibitory effect of LEN on the expression of c-maf, USP7, and FTH11. Figure 43 Furthermore, Western blot analysis showed that, compared with the control group, LEN treatment significantly reduced the expression of c-Maf, USP7, and FTH1, while co-treatment with LEN and DFO significantly reversed the inhibitory effect of LEN on the expression of c-maf, USP7, and FTH1. Figure 44 In summary, these results indicate that LEN induces ferroptosis in vivo via the c-Maf / USP7 / FTH1 signaling pathway.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a reagent that downregulates FTH1 expression in the preparation of a drug for treating multiple myeloma.

2. The application according to claim 1, characterized in that, The reagent induces ferroptosis by adjusting the c-Maf / USP7 / FTH1 axis.

3. The application according to claim 2, characterized in that, The reagent has at least one of the following functions: binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

4. The application according to claim 1, characterized in that, The reagents include lenalidomide.

5. The application of an ferroptosis-inducing agent in the preparation of a drug for treating multiple myeloma.

6. The application according to claim 5, characterized in that, The ferroptosis includes ferroptosis induced by the c-Maf / USP7 / FTH1 axis.

7. The application according to claim 5, characterized in that, The reagent has at least one of the following functions: binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

8. The application according to claim 7, characterized in that, The reagents include lenalidomide.

9. A drug for treating multiple myeloma, characterized in that, The active ingredient of the drug has at least the following effects: inducing ferroptosis, binding c-Maf, blocking c-Maf-mediated USP7 transcription, inhibiting USP7 expression, and downregulating FTH1 expression.

10. The drug according to claim 9, characterized in that, The active ingredient of the drug includes lenalidomide.

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