Molecular glue application of mitoxantrone in inducing irf4 protein degradation via autophagy-lysosomal pathway

Abstract

The application of mitoxantrone in inducing IRF4 protein degradation through autophagy-lysosome pathway belongs to the field of biological medicine. Mitoxantrone can specifically bind to IRF4 protein and significantly reduce the level of IRF4 protein. Mitoxantrone mainly binds to the DNA binding domain of IRF4, shortens the half-life of IRF4 protein, and promotes its degradation through the autophagy-lysosome pathway rather than the proteasome pathway. Mitoxantrone can interact with IRF4 protein and autophagy receptor p62 at the same time, promote the formation of IRF4 and p62 complex, enhance the ubiquitination modification of IRF4 and recruit it to the autophagy degradation pathway, thereby playing the role of molecular glue. Mitoxantrone treatment can inhibit the transcriptional activation function mediated by IRF4, down-regulate the expression of its downstream target genes, inhibit the proliferation of multiple myeloma cells and tumor growth, and provide a new technical approach for the development of anti-tumor drugs targeting transcription factors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a molecular glue-like strategy that targets the autophagy-lysosomal pathway to regulate the stability of the transcription factor IRF4 protein, and particularly to the novel use of mitoxantrone in inducing selective autophagic degradation of the IRF4 protein. Background Technology

[0002] Precise regulation of protein homeostasis is crucial for maintaining normal cellular function. Two main protein degradation systems exist in eukaryotic cells: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system. The UPS primarily mediates the degradation of short-lived, soluble proteins, while the autophagy-lysosome pathway plays a key role in clearing long-lived proteins, protein aggregates, and specific functional proteins.

[0003] Selective autophagy is a protein degradation pathway that has attracted widespread attention in recent years. Its core characteristic lies in the ability of autophagy receptor proteins (such as p62 / SQSTM1) to recognize ubiquitinated substrate proteins and recruit them to autophagosomes via the LC3 interaction domain (LIR), ultimately leading to degradation in lysosomes. Compared to UPS, the autophagy-lysosomal pathway has unique advantages in degrading "non-classical druggable targets" such as transcription factors and signal transduction proteins.

[0004] IRF4 (Interferon Regulatory Factor 4) is an immune-related transcription factor that plays a central regulatory role in plasma cell differentiation and specific transcriptional programs in tumor cells. IRF4 drives the transcription of various downstream genes through its DNA-binding domain (DBD) and IRF-binding domain (IAD). However, due to the lack of a clear enzymatic activity pocket and its high structural flexibility, IRF4 has long been considered an "untreatable" target.

[0005] In recent years, the molecular glue strategy has provided a new approach for drug development targeting "undruggable" targets by inducing protein-protein interactions through small molecules, thereby achieving target protein degradation. Therefore, developing a novel small molecule strategy that can target IRF4 and induce its degradation through the autophagy-lysosomal pathway has significant theoretical implications and potential application value. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems in the prior art and provide a molecular glue-like technology solution for inducing the degradation of transcription factor IRF4 protein through the autophagy-lysosomal pathway. Specifically, it involves the new use of mitoxantrone (MIT) in regulating the stability of IRF4 protein, thereby providing a new intervention strategy for transcription factors that are difficult to target directly.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The use of mitoxantrone in the preparation of drugs that regulate the stability of the transcription factor IRF4 protein, said use being achieved by promoting the degradation of IRF4 protein via the autophagy-lysosomal pathway.

[0009] The mitoxantrone can bind directly to the IRF4 protein, thereby reducing the stability of the IRF4 protein.

[0010] The mitoxantrone interacts with the DNA-binding domain of the IRF4 protein, thereby affecting the stability and functional activity of the IRF4 protein.

[0011] The mitoxantrone promotes the interaction between the IRF4 protein and the autophagy receptor p62 protein.

[0012] The mitoxantrone can bind to both IRF4 and p62 proteins simultaneously to form an IRF4-mitoxantrone-p62 ternary complex, thereby acting as a molecular glue to recruit the IRF4 protein to the autophagy-lysosomal degradation pathway.

[0013] The application described herein can inhibit IRF4-mediated transcriptional activation; the transcriptional activation includes inhibition of transcriptional activation functions selected from... c Regulation of the expression of at least one IRF4 downstream target gene among -MYC, KLF2 and LDHA.

[0014] The application is capable of inhibiting the proliferation of IRF4-dependent tumor cells; the tumor cells are multiple myeloma cells with high IRF4 expression.

[0015] A pharmaceutical composition comprising mitoxantrone or a pharmaceutically acceptable carrier or excipient thereof, the pharmaceutical composition being used to target the autophagy-lysosomal degradation of IRF4.

[0016] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0017] 1. This invention provides a novel mechanism of action that differs from the traditional application of mitoxantrone. It promotes the degradation of IRF4 protein via autophagy-lysosome pathway through a molecular gel-like approach, thereby effectively regulating IRF4-related biological processes and providing a new technical path for drug development targeting untreatable transcription factors.

[0018] 2. Treatment with mitoxantrone can reduce the expression level of IRF4 protein and inhibit the transcriptional activity of downstream genes mediated by IRF4; at the same time, treatment with mitoxantrone can accelerate the degradation process of IRF4 protein, and this degradation mainly depends on the autophagy-lysosomal pathway rather than the proteasome pathway.

[0019] 3. Mitoxantrone exerts its effects by regulating the stability of the IRF4 protein, including at least one of promoting IRF4 protein degradation, inhibiting IRF4 protein functional activity, and reducing IRF4-mediated downstream transcriptional activity, thereby affecting IRF4-related cell biological processes.

[0020] 4. The mitoxantrone-induced IRF4 protein degradation mainly depends on the autophagy-lysosomal pathway, specifically including at least one of promoting the interaction between IRF4 and the autophagy receptor protein p62, enhancing IRF4 entry into the selective autophagy degradation pathway, and accelerating IRF4 protein clearance. Attached Figure Description

[0021] Figure 1 Mitoxantrone (MIT), a molecular gel targeting IRF4, was identified using high-throughput virtual screening. Figure 1 A represents a high-throughput virtual screening workflow for inhibitors targeting human IRF4 protein; Figure 1 B represents the chemical structure of MIT and the results of its computer-generated molecular docking with the active pocket of the human IRF4 protein. Figure 1 In the middle, C represents the cellular thermal shift assay (CETSA) used to detect changes in the thermal stability of IRF4 protein in ARP-1 or IM-9 cells after MIT treatment; Figure 1 The diagram in D shows the construction of the IRF4 truncated form, which includes a fragment lacking the DNA-binding domain (ΔDBD) and the IRF-associated domain (ΔIAD). Figure 1 E represents the expression of different IRF4 truncated variants (ΔDBD and ΔIAD) in HEK293T cells by Western blot analysis. Figure 1 In the middle F, CETSA was used to detect changes in the thermostability of HEK293T cells transfected with different IRF4 truncated variants (ΔDBD and ΔIAD) plasmids after MIT treatment or without treatment. Figure 1 The diagram in G represents the mutation site of the IRF4 protein; Figure 1 In the middle, H represents the expression of IRF4 protein in HEK293T cells after GFP ligation, detected by Western blot. Figure 1 In the middle, I represents the microscale thermophoresis (MST) experiment used to detect changes in the binding ability of MIT with wild-type (WT) and mutant (Mut) IRF4.

[0022] Figure 2 This is a diagram illustrating the effect of MIT in reducing IRF4 protein expression and inhibiting its downstream transcriptional activity. (The diagram shows the effect of MIT in reducing IRF4 protein expression and inhibiting its downstream transcriptional activity.) Figure 2In the figure, A represents the IRF4 protein expression level in ARP-1 or IM-9 cells after MIT treatment or without treatment; Figure 2 In section B, ARP-1 or IM-9 cells were treated with MIT or not. c - Relative mRNA expression levels of MYC, KLF2, and LDHA; Figure 2 In the middle, C represents the ChIP-PCR detection of IRF4 in ARP-1 or IM-9 cells after MIT treatment or without treatment. c - Enrichment on the MYC promoter; Figure 2 In the experiment, D represents cycloheximide (CHX, 100 μM) to detect the time-dependent changes in IRF4 protein stability in ARP-1 or IM-9 cells after CHX treatment with or without MIT. Figure 2 E represents the expression level of IRF4 protein in ARP-1 or IM-9 cells after treatment with the proteasome inhibitor MG-132 or the lysosomal inhibitor Leupeptin, with or without MIT treatment.

[0023] Figure 3 This diagram illustrates the role of MIT as a molecular gel-like substance in promoting the degradation of IRF4 via the autophagy-lysosomal pathway. Among other things, Figure 3 In the middle section, A represents the expression levels of IRF4, p62, and LC3B proteins and the analysis of co-immunoprecipitation (Co-IP) in ARP-1 or IM-9 cells treated with chloroquine (CQ) with or without MIT. Figure 3 In the study, ARP-1 or IM-9 cells were treated with or untreated with MIT, and immunofluorescence staining was performed using DAPI, IRF4, and p62 antibodies. Figure 3 In the middle (C), C represents the change in the thermostability of p62 protein in ARP-1 or IM-9 cells after MIT treatment, detected by CETSA. Figure 3 In the middle section, D represents the isothermal dose-response cellular thermal shift assay (ITDRF-CETSA) to detect changes in the thermal stability of p62 protein in ARP-1 or IM-9 cells after treatment with different doses of MIT at 25 ℃ and 52 ℃. Figure 3 The left side of E in the middle section shows a computer simulation of the binding conformation of the MIT-IRF4 / p62 ternary complex; Figure 3 The right side of the middle E represents the specific combination mode of MIT.

[0024] Figure 4 This image shows the effect of MIT in inhibiting the proliferation of multiple myeloma cells. Among them, Figure 4Image A shows representative images of ARP-1 cells after MIT treatment or without EdU staining, and the proportion of EdU-positive cells. Figure 4 B represents the cell proliferation curves of ARP-1 cells after treatment with different concentrations of MIT; Figure 4 In the middle (C), there are representative images of IM-9 cells after MIT treatment or without MIT staining, and the proportion of EdU-positive cells. Figure 4 D represents the cell proliferation curves of IM-9 cells after treatment with different concentrations of MIT; Figure 4 E represents the cell viability of LP-1 cells after MIT treatment; Figure 4 In the middle F, the cell viability of H929 cells after MIT treatment is shown. Figure 4 G represents the cell viability of RPMI 8226 cells after MIT treatment; Figure 4 In the middle, H represents the cell viability of MM.1S cells after MIT treatment; Figure 4 In Figure I, qPCR analysis was used to determine the relative mRNA expression level of IRF4 in the IRF4-overexpressing cell line (ARP-1). Figure 4 J represents Western blot analysis of IRF4 protein expression levels in the IRF4-overexpressing cell line (ARP-1); Figure 4 In the cytotoxicity assay, K represents the half-maximal inhibitory concentration (IC50) of MIT against ARP-1 cells. Figure 4 In the cytotoxicity assay, L represents the IC50 of MIT against the IRF4-overexpressing cell line (ARP-1). Figure 4 In the middle section, M represents the qPCR analysis of the relative mRNA expression level of IRF4 in the IRF4-overexpressing cell line (IM-9); Figure 4 In the middle, N represents the IRF4 protein expression level in the IRF4-overexpressing cell line (IM-9) analyzed by Western blot. Figure 4 In the cytotoxicity assay, MIT was used to determine the IC50 of IM-9 cells. Figure 4 In the cytotoxicity assay, P represents the IC50 of MIT against the IRF4-overexpressing cell line (IM-9).

[0025] Figure 5 This image shows the effect of MIT in inducing IRF4 protein degradation and inhibiting tumor-related phenotypes in an animal model of multiple myeloma. Figure 5 In the image A, there are representative photos of mouse tumors after NSG mice were subcutaneously inoculated with ARP-1 or IM-9 cells to establish a xenograft model, and then treated with MIT intraperitoneal injection or a vector control. Figure 5 In the middle B, the tumor weight change in NSG mice is shown. Figure 5 In the middle, C represents the tumor volume change in NSG mice; Figure 5 D represents tumor tissue in NSG micec - Relative expression levels of MYC, KLF2, and LDHA; Figure 5 E represents the immunofluorescence staining results of IRF4 in tumor tissue; Figure 5 In the middle F, the results of immunofluorescence staining of Ki67 in tumor tissue are shown. Figure 5 G represents the expression level of IRF4 protein in NSG mouse tumor tissue; Figure 5 H is Expression level of IRF4 protein in mouse spleen cells; Figure 5 I is Representative images of mouse spleen tumor cells stained with DAPI, CD138, and IRF4 antibodies using immunofluorescence. Figure 5 C57BL / 6J mice were inoculated with J. Representative photographs of the spleens of mice treated with or without intraperitoneal injection of MIT after establishing a myeloma model using cells. Figure 5 K in the middle represents ELISA detection. Concentration of IgG2b in mouse serum; Figure 5 The diagram in L represents the mechanism by which MIT regulates IRF4 degradation. Detailed Implementation

[0026] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0027] Example 1: Identification of the interaction between mitoxantrone (MIT) and IRF4 protein

[0028] 1. Test Methods

[0029] 1.1 Cellular Thermal Shift Assay (CETSA) Method

[0030] In this embodiment, the CETSA method was used to detect the interaction between MIT and IRF4 protein / truncated variant. Multiple myeloma (MM) cells, after appropriate treatment, were collected, washed with pre-cooled buffer, and resuspended in a suitable buffer system. The cell samples were then aliquoted and subjected to heat treatment under different heating conditions to induce differences in the thermal stability of intracellular proteins. After heat treatment, the samples were immediately cooled to terminate the thermal denaturation process. The cooled cell samples were lysed with cell lysis buffer, incubated at low temperature, and then centrifuged to obtain soluble protein components. The obtained protein samples were quantified, and the protein concentrations of each sample were adjusted to a consistent level.

[0031] Subsequently, protein denaturation buffer was added to each sample, and after heating and denaturation, polyacrylamide gel electrophoresis was performed to separate the proteins, which were then transferred to a membrane. After blocking, specific antibodies against the target protein and corresponding secondary antibodies were added for incubation, and the protein signal intensity was detected by chemiluminescence. By comparing the residual levels of the target protein under different treatment conditions and different heating conditions, the effect of MIT treatment on the thermostability of IRF4 protein was evaluated, thereby determining whether there is an interaction between MIT and IRF4 protein.

[0032] 1.2 Microscale Thermophoresis (MST) Analysis

[0033] In this embodiment, the MST (Medium-Stage Thermogravimetric Analysis) technique was used to detect the binding ability between MIT and IRF4 protein / IRF4 protein post-point mutation. The purified IRF4 protein or its functional fragment was placed in a suitable buffer system, and the protein was detected using its intrinsic fluorescence signal. Subsequently, different concentrations of MIT were mixed with the protein samples and incubated under suitable conditions to reach binding equilibrium. The incubated samples were loaded into capillaries and detected using a micro-thermogravimetric detection system. By applying a local temperature gradient, changes in the thermophoretic behavior of the protein-small molecule complex were induced. By recording and analyzing the changes in the protein thermophoretic signal under different MIT concentrations, binding curves between the protein and MIT were obtained. Based on the binding curves, the binding parameters between MIT and the protein were calculated to assess their binding affinity. The detection results under different treatment conditions or different protein constructs were compared to further confirm the specific binding relationship between MIT and IRF4 protein.

[0034] 2. Test Results

[0035] 2.1. Screening of compound libraries and the discovery at MIT

[0036] To find molecular gels that can target IRF4, we conducted a high-throughput virtual screening based on the IRF4 protein structure. Figure 1 (A) We screened a compound library of 24,600 compounds to search for small molecules that might bind to IRF4. The screening employed a stepwise docking strategy, first performing standard-precision molecular docking, followed by high-precision molecular docking, from which the candidate compound MIT was obtained. Molecular docking results showed that MIT can form 6 hydrogen bonds and 2 salt bridges with IRF4, acting on four amino acid residues: Gly39, Glu66, Leu70, and Asp120. Figure 1 (B)

[0037] 2.2 Verification of the interaction between MIT and IRF4 protein

[0038] To verify whether MIT can directly bind to IRF4, we first used CETSA to detect the interaction between MIT and IRF4 in ARP-1 and IM-9 MM cells. The results showed that MIT and IRF4 bind stably. Figure 1 (C)

[0039] Furthermore, previous studies have shown that IRF4 contains two highly conserved domains: a DNA-binding domain (ΔDBD) located at the N-terminus and an IRF-associated domain (ΔIAD) located at the C-terminus. Figure 1 To clarify the binding region between MIT and IRF4, we constructed IRF4 truncated mutants with missing DBD (ΔDBD) and missing IAD (ΔIAD), respectively. Figure 1 (E). The interaction between the IRF4-ΔDBD / ΔIAD domain and MIT was compared using CETSA analysis, confirming that MIT primarily binds to the DNA-binding domain of the IRF4 protein. Figure 1 (Middle F).

[0040] To further determine the specific binding site, we mutated Glu66 and Leu70 to alanine ( Figure 1 (G). Western blot was used to detect IRF4 and IRF4 mutant proteins linked to GFP fluorescent protein (G). Figure 1 The binding affinity was then assessed using MST analysis. The results showed that the above mutation significantly weakened the binding affinity between MIT and IRF4. Figure 1 (I). The above results indicate that MIT can directly bind to IRF4, primarily depending on the Glu66 and Leu70 sites.

[0041] Example 2: Effects of MIT on IRF4 protein stability and transcriptional function

[0042] 1. Test Methods

[0043] 1.1 Method for analyzing the half-life of cycloheximide (CHX) protein

[0044] In this embodiment, a protein synthesis inhibition assay was used to detect the effect of MIT on the stability of IRF4 protein. After MM cells were cultured to an appropriate density, the protein synthesis inhibitor CHX was added to block the synthesis of nascent protein, while MIT treatment was administered with or without administration. Cell samples were collected at different time points, and the cells were lysed using a suitable lysis buffer to obtain total protein.

[0045] After quantitative analysis of the protein samples, the expression level of IRF4 protein was detected by Western blotting. By comparing the decay of IRF4 protein at different time points, the effect of MIT treatment on the half-life of IRF4 protein was evaluated, thereby determining its regulatory role in the stability of IRF4 protein.

[0046] 1.2 Method for detecting IRF4-mediated transcriptional activity of downstream target genes using real-time quantitative PCR (qPCR)

[0047] In this embodiment, qPCR was used to detect the effect of MIT on the transcriptional activity of IRF4 downstream target genes. MM cells were treated with MIT under suitable conditions, and the cells were collected and total RNA was extracted. The RNA was reverse transcribed to generate cDNA, which was then used as a template for real-time quantitative PCR amplification to detect the transcriptional levels of IRF4 downstream target genes. By using an internal reference gene as a standard, the relative expression levels of target genes in each treatment group were analyzed and compared to evaluate the effect of MIT on regulating IRF4 protein levels and the transcriptional activity of its downstream target genes.

[0048] 1.3 Chromatin Immunoprecipitation (ChIP) Experimental Analysis Method

[0049] In this embodiment, the effect of MIT on the binding ability of IRF4 to the promoter region of target genes was detected using a ChIP assay. After appropriate cell treatment, the protein and DNA were cross-linked and fixed using a cross-linking agent, followed by termination of the cross-linking reaction and cell lysis. The resulting chromatin was fragmented to obtain DNA fragments of suitable length. The chromatin sample was immunoprecipitated with a specific antibody against IRF4, with IgG used as a control. After immunoprecipitation, the bound DNA-protein complex was eluted and the cross-linking was removed, and DNA was extracted. Real-time quantitative PCR was used to detect the enrichment of the target gene promoter region. By comparing the binding level of IRF4 in the target gene promoter region under different treatment conditions, the effect of MIT on the transcriptional regulatory function of IRF4 was evaluated.

[0050] 1.4 Treatment methods for autophagy inhibitors and proteasome inhibitors

[0051] In this embodiment, to clarify the IRF4 protein degradation pathway, cells were treated with autophagy inhibitors and proteasome inhibitors, respectively. MM cells were cultured under suitable conditions and treated with MIT (mitogen-induced degradation) in combination with either an autophagy inhibitor or a proteasome inhibitor. After treatment, cells were collected and total protein was extracted. Changes in IRF4 protein expression were detected by Western blotting analysis, and the differences in IRF4 protein levels under different inhibitor treatments were compared to determine the main pathway underlying MIT-induced IRF4 protein degradation.

[0052] 2. Test Results

[0053] 2.1 Effects of MIT treatment on IRF4 protein expression levels and function

[0054] Functional validation revealed that MIT significantly reduced the expression level of IRF4 protein in MM cells. Figure 2 (A), while significantly inhibiting its downstream. c - The relative mRNA expression levels of target genes such as MYC, KLF2, and LDHA ( Figure 2 (B). Furthermore, MIT significantly reduced IRF4 levels. c - Enrichment level of the MYC starter subregion ( Figure 2 (C)

[0055] 2.2 Effect of MIT on IRF4 protein stability

[0056] We used the protein synthesis inhibitor CHX to track the stability of the IRF4 protein. The results showed that MIT treatment significantly accelerated the degradation rate of the IRF4 protein compared to the control group. Figure 2 (D) suggests that MIT can promote the degradation of IRF4 protein.

[0057] 2.3 Mechanism of action of MIT in degrading IRF4 protein

[0058] Given that the proteasome and lysosome are the two main degradation pathways for maintaining protein homeostasis, we treated cells with the proteasome inhibitor MG-132 and the lysosomal protease inhibitor Leupeptin, respectively. The results showed that only Leupeptin could reverse the MIT-induced decrease in IRF4 protein, while MG-132 did not produce the same effect. Figure 2 The results (E) indicate that MIT promotes IRF4 degradation mainly through the autophagy-lysosome pathway.

[0059] Example 3: MIT-mediated molecular glue-like mechanism and autophagy degradation process

[0060] 1. Test Methods

[0061] 1.1 Co-IP combined with immunoblotting analysis and ubiquitination detection method

[0062] MM cells were cultured under standard conditions and treated with MIT according to the experimental design. Under some experimental conditions, treatment with autophagy inhibitors was performed to enhance the accumulation of ubiquitinated proteins or interacting complexes. After treatment, cells were collected and lysed using lysis buffer containing protease inhibitors to obtain total cell protein lysate.

[0063] The resulting lysate was centrifuged, and the supernatant was used as the starting sample for immunoprecipitation. A portion of the lysate was used as input for subsequent immunoblotting analysis, while the remaining sample was used for IRF4-p62 interaction detection and IRF4 protein ubiquitination level detection, respectively.

[0064] In the IRF4-p62 interaction assay, the protein lysis buffer was incubated with a specific antibody against IRF4 or p62 to form an immune complex, with an isotype control antibody used as a negative control. Subsequently, a protein G carrier was added to capture the immune complex. After incubation and multiple washes, loading buffer was added and the mixture was heated to dissociate the protein complex from the carrier.

[0065] In the detection of IRF4 protein ubiquitination, a similar immunoprecipitation procedure was used, with IRF4 as the target protein for immunoprecipitation, to obtain enriched IRF4 protein complexes. The obtained samples were separated by electrophoresis and transferred to a membrane, and then analyzed by Western blotting using a specific antibody against ubiquitin to detect the ubiquitination modification level of the IRF4 protein.

[0066] 1.2 Immunofluorescence (IF) Detection

[0067] MM cells were seeded onto culture vectors suitable for IF (influence-related protein) observation. After stable cell growth, they were treated with MIT (mitotically active protein) according to the experimental design; a control group without treatment was also included. After treatment, the cells were fixed and permeabilized to maintain the spatial distribution of intracellular proteins. Subsequently, the cells were blocked using blocking buffer to reduce non-specific binding. After blocking, specific primary antibodies against IRF4 and p62 were added and incubated to allow the antibodies to fully bind to their corresponding target proteins. After washing to remove unbound antibodies, secondary antibodies labeled with different fluorescent dyes were added and incubated to distinguish the fluorescence signals of IRF4 and p62. The cell nuclei were counterstained using nuclear dyes. After staining, the cells were observed and imaged using laser confocal microscopy, and the distribution of IRF4 and p62 fluorescence signals was analyzed. The co-localization level of IRF4 and p62 in cells was assessed by comparing the degree of overlap of the two fluorescence signals in cells from different treatment groups.

[0068] 2. Test Results

[0069] 2.1 Detection of the interaction between MIT and IRF4 and autophagy receptor p62.

[0070] To elucidate the molecular mechanism by which MIT induces IRF4 protein degradation, the role of autophagy receptor proteins in this process was further investigated. Western blot results showed that, compared with the control group, MIT treatment significantly enhanced the interaction between IRF4 and p62, and simultaneously increased the ubiquitination level of IRF4 protein. Figure 3 (A) Combined with the results under autophagy inhibitor treatment, it can be further confirmed that MIT accelerates the ubiquitination of IRF4 protein by promoting the binding of IRF4 to p62, and recruits it to the autophagy-lysosomal degradation pathway. This indicates that MIT can promote the formation of a stable complex between IRF4 and the autophagy receptor p62, induce IRF4 protein ubiquitination, and accelerate its degradation, providing experimental evidence for its role as a molecular glue-like regulator of IRF4 protein stability.

[0071] The IF experiment results showed that, compared with the control group, MIT treatment significantly enhanced the co-localization signal of IRF4 and p62 in MM cells, manifested as increased overlap of the two fluorescent signals in the same cellular region. Figure 3 (B) Combined with the aforementioned immunoprecipitation and ubiquitination results, it is shown that MIT can promote the spatial aggregation and interaction of IRF4 and p62 at the cellular level, thereby facilitating the recruitment of IRF4 protein to autophagy-related degradation pathways.

[0072] 2.2 Verification of the interaction between MIT and p62 protein

[0073] To further verify whether MIT can directly bind to p62, we also used the CETSA experiment to detect the interaction between MIT and p62. The results showed that MIT can significantly enhance the thermal aggregation of p62 protein. Figure 3 (C). Subsequently, isothermal dose-dependent CETSA reactions were performed at 25℃ and 52℃. The experimental results showed that MIT can promote the thermally induced aggregation of p62 protein in a dose-dependent manner. Figure 3 (D). The results indicate that MIT can interact with the p62 protein.

[0074] 2.3 Verification of the MIT-like molecular glue interaction between IRF4 and p62 proteins

[0075] The interaction between MIT and the IRF4 protein and the autophagy receptor p62 was further investigated using molecular docking analysis. Molecular docking results showed that MIT can not only form a stable binding with four amino acid residues of IRF4 (Gly39, Glu66, Leu70, and Asp120), but also bind to the Leu400 site on the p62 protein. Figure 3 (E), thus forming a stable ternary complex conformation.

[0076] Example 4: Effects of MIT on MM cell proliferation

[0077] 1. Test Methods

[0078] 1.1 EdU method for detecting cell proliferation capacity

[0079] MM cells were seeded in a suitable culture system. When the cells were in the logarithmic growth phase, different doses of MIT were administered according to the experimental design, with a corresponding untreated control group. After a certain treatment time, EdU-labeled reagent was added to the culture system to incorporate into newly synthesized nucleic acid chains during DNA synthesis. Subsequently, the cells were fixed, permeabilized, and fluorescently labeled. EdU-positive cells were observed and statistically analyzed using a fluorescence microscope. The effect of MIT on DNA synthesis and proliferation of MM cells was evaluated by comparing the proportion of EdU-positive cells in different treatment groups.

[0080] 1.2. CCK-8 assay for cell viability and toxicity

[0081] To further evaluate the effects of MIT on cell viability and potential cytotoxicity, MM cells were seeded in 96-well culture plates and treated with different concentrations of MIT after stable cell growth; a blank control group was also included. After a certain treatment time, cell viability assay reagents were added to each well, and the cells were incubated in the dark to allow the dehydrogenases in the live cells to react with the assay reagents. Subsequently, the cell viability of each treatment group was calculated by measuring the absorbance values ​​at the corresponding wavelengths.

[0082] 1.3 Determination and Analysis of IC50

[0083] Based on the above CCK-8 assay, dose-response curves were plotted according to the changes in cell viability after treatment with different concentrations of MIT, and the half-maximal inhibitory concentration (IC50) of MIT on MM cells was calculated by nonlinear regression analysis.

[0084] 2. Test Results

[0085] 2.1 MIT's drug efficacy test results

[0086] Given the central role of IRF4 in the development and progression of multiple myeloma (MM), we further evaluated the effect of MIT on MM growth. Experimental results showed that, compared with the control group, MIT treatment significantly increased ARP-1 levels (…). Figure 4 (A) and IM-9 ( Figure 4 The proportion of EdU-positive cells in C cells was significantly reduced. Cell proliferation experiments showed that MIT could significantly inhibit ARP-1 (… Figure 4 (B) and IM-9 ( Figure 4 The proliferation of D) cells. Furthermore, MIT significantly inhibited LP-1 ( Figure 4 (E), H929 ( Figure 4 (China F), RPMI 8226 ( Figure 4 (G) and MM.1s ( Figure 4 The compound exhibited activity against various MM cells, indicating that it has a strong inhibitory effect on MM cells.

[0087] 2.2 Validation of the inhibitory effect of MIT on IRF4-dependent cell proliferation

[0088] Validated by qPCR and Western blot, we found that ARP-1 ( Figure 4 China I, J) and IM-9 ( Figure 4 IRF4 protein was overexpressed in M ​​and N cells. The results showed that IRF4-high expression of ARP-1 (… Figure 4 (K, L) and IM-9 ( Figure 4 O and P cells were more sensitive to MIT treatment, suggesting that the inhibitory effect of MIT on cell proliferation is closely related to its induction of IRF4 protein degradation.

[0089] Example 5: Effects of MIT in animal models

[0090] 1. Test Methods

[0091] 1.1 Methods for detecting drug efficacy in immunodeficient mouse xenotransplantation models

[0092] Immunodeficient mice were selected as experimental animals, and MM cell lines were subcutaneously injected into the mice to construct an MM xenograft model. After tumor formation and reaching a stable growth stage, the model mice were treated with drugs starting approximately two weeks after cell inoculation. According to the experimental design, MIT was administered via intraperitoneal injection twice weekly for two weeks; a corresponding control group was also included. During the drug administration period, subcutaneous tumors in the mice were measured regularly to record changes in tumor volume and assess the effect of MIT on tumor growth. After drug administration, the experimental animals were euthanized, and the tumor tissue was dissected and weighed. Simultaneously, molecular biological analysis was performed on the obtained tumor tissue, including extraction of nucleic acids from the tumor tissue and detection of the expression levels of IRF4 and its downstream target genes, as well as related pathological section analysis, to evaluate the regulatory role of MIT on IRF4-related signaling pathways in vivo.

[0093] 1.2 Methods for detecting drug efficacy in immunocompetent MM mouse models

[0094] To further evaluate the therapeutic effect of MIT on MM in the context of intact immune function, immunocompetent mice were selected as experimental animals. The MM cell line (vk12598) was injected into mice via tail vein injection to establish a systemic MM model. Approximately one week after cell injection, the model mice underwent mitogenic treatment (MIT) via intraperitoneal injection, twice weekly for three weeks. A control group was also included. Growth status of the mice was observed during the treatment period. After the experiment, the mice were processed and relevant samples were collected. Splenomegaly was assessed by visual observation and measurement of spleen size. Simultaneously, peripheral blood samples were collected via tail vein to detect MM-related markers in serum, evaluating the effect of MIT on tumor burden.

[0095] 1.3 Enzyme-linked immunosorbent assay (ELISA) method

[0096] To further evaluate the impact of mitochondrial tumor burden (MIT) on tumor burden in multifocal tumors (MM), the level of M protein in the serum of model mice was detected and analyzed using an ELISA method. At the end of the animal experiment, peripheral blood samples were collected from mice via tail vein sampling. After standing and centrifugation, serum samples were obtained. The obtained serum samples were appropriately diluted according to the experimental design and added to ELISA reaction wells pre-coated with antibodies targeting immunoglobulins or their specific light chains, allowing the M protein in the serum to specifically bind to the coated antibodies. After the binding reaction was completed and unbound components were washed away, enzyme-labeled detection antibodies were added for incubation, forming antibody-antigen-antibody immune complexes. Subsequently, a chromogenic substrate was added to induce a detectable color change in the enzymatic reaction. After the reaction was terminated, the relative content of M protein in the serum was reflected by detecting the absorbance value at the corresponding wavelength. The changes in the level of M protein in the serum of mice in each experimental group were calculated by comparing with a standard curve or a control group.

[0097] 2. Test Results

[0098] 2.1 Verification of the effects of MIT in vivo

[0099] We established a subcutaneous xenograft tumor model using ARP-1 and IM-9 cells in NSG mice to evaluate the in vivo antitumor effect of MIT. After MIT treatment for a period of time, tumor tissue and related indicators were analyzed. Results showed that MIT treatment significantly reduced tumor progression in the immunodeficient mouse xenograft model. Figure 5 In the middle A), the tumor weight was significantly reduced ( Figure 5 (B) The volume growth slowed significantly ( Figure 5 In the middle C, the expression of the tumor marker Ki67 was significantly reduced ( Figure 5 (Middle F).

[0100] To further validate the therapeutic effect of MIT in an immune integrity model, we used... Mouse model. Cells were injected into C57BL / 6J mice via tail vein injection, followed by MIT treatment starting two weeks later and continuing for two weeks. Results showed that MIT treatment restored spleen size in immunocompetent MM mouse models. Figure 5 In the middle J), ​​serum M protein expression was significantly decreased ( Figure 5 In the middle K, the expression of the tumor marker CD138 in the spleen was significantly decreased ( Figure 5 Middle I).

[0101] 2.2 Study on the regulatory role of MIT on the expression of IRF4 and its downstream target genes in an in vivo model

[0102] IF ( Figure 5 (E) and Western blot ( Figure 5 Further analysis using the G10 assay confirmed that MIT significantly reduced the expression level of IRF4 protein in vivo. Simultaneously, downstream IRF4... c The expression levels of target genes MYC, KLF2, and LDHA were also significantly downregulated. Figure 5 The results (D) suggest that MIT inhibits IRF4-mediated transcriptional activation under in vivo conditions.

[0103] In addition, in individuals with normal immune function In a mouse model, Western blot analysis was performed on spleen tissue. Figure 5 H) and IF analysis ( Figure 5 The study found that MIT administration significantly inhibited the expression level of IRF4 protein in the spleen, indicating that the compound can also regulate IRF4-related signaling pathways in the context of intact immunity.

[0104] The results of the above examples indicate that MIT can act as a molecular glue to mediate the binding of IRF4 and p62, thereby promoting the degradation of IRF4 via the autophagy-lysosomal pathway and ultimately significantly inhibiting MM growth in vitro and in vivo. Figure 5 (L). The technical solution provided by this invention offers a new technical basis for the development of IRF4-targeted regulation and molecular-like gel drugs.

Claims

1. The use of mitoxantrone in the preparation of drugs that regulate the stability of transcription factor IRF4 protein, characterized in that: The intended use is achieved by promoting the degradation of IRF4 protein via the autophagy-lysosomal pathway.

2. The use as described in claim 1, characterized in that: The mitoxantrone can bind directly to the IRF4 protein, reducing the stability of the IRF4 protein.

3. The use as described in claim 2, characterized in that: The mitoxantrone interacts with the DNA-binding domain of the IRF4 protein.

4. The use as described in claim 1, characterized in that: The mitoxantrone promotes the interaction between the IRF4 protein and the autophagy receptor p62 protein.

5. The use as described in claim 4, characterized in that: The mitoxantrone can bind to both IRF4 and p62 proteins simultaneously to form an IRF4-mitoxantrone-p62 ternary complex, thereby acting as a molecular glue to recruit the IRF4 protein to the autophagy-lysosomal degradation pathway.

6. The use as described in any one of claims 1 to 5, characterized in that: The application described herein can inhibit IRF4-mediated transcriptional activation; the transcriptional activation includes inhibition of transcriptional activation functions selected from... c Regulation of the expression of at least one IRF4 downstream target gene among -MYC, KLF2 and LDHA.

7. The use as described in any one of claims 1 to 5, characterized in that: The application is capable of inhibiting the proliferation of IRF4-dependent tumor cells; the tumor cells are multiple myeloma cells with high IRF4 expression.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises mitoxantrone or a pharmaceutically acceptable carrier or excipient thereof, and the pharmaceutical composition is used to target the autophagy-lysosomal degradation of IRF4.

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