Application of 4.1 R protein as multiple myeloma CD40 / NF-kappa B signaling pathway inhibitor and drug

Abstract

The invention relates to application of 4.1 R protein as a multiple myeloma CD40 / NF-kappa B signal channel inhibitor and a drug, and belongs to the technical field of biological medicine. According to the present invention, the FERM and CTD structural domains of the 4, 4, 1 R interact with CD40, and form a compound with NF-kappa B p50 so as to act as the scaffold protein in the CD40 / NF-kappa B axis, inhibit abnormal NF-kappa B activation, further inhibit the CD40 / NF-kappa B signaling pathway, and provide the anti-cancer effect;

Description

Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to the application of 4.1R protein as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma and related drugs. Background Technology

[0003] Multiple myeloma (MM) is a hematologic malignancy characterized by the clonal expansion of malignant plasma cells in the bone marrow, accounting for approximately 10% of all hematologic cancers. It is generally believed that most MM cases originate from undetermined monoclonal gammopathy of undetermined significance (MGUS), subsequently evolving into smoking multiple myeloma (SMM) through the accumulation of genetic damage and clonal selection, eventually progressing to symptomatic MM. During this process, repeated mutations in key genes such as MYC, KRAS, NRAS, BRAF, and TP53 drive alterations in cell proliferation, apoptosis, and signaling pathways. Although the CD40 / NF-κB axis is widely recognized as the central pathway for maintaining MM cell survival, its upstream regulatory mechanisms remain incompletely understood. Aberrant NF-κB activation is a hallmark of MM pathogenesis, driven by both intrinsic genetic alterations and extrinsic signals from the bone marrow microenvironment, such as pro-inflammatory cytokines like TNF-α and IL-1β. Sustained activation of both the classical (IKK / IκB-p50 / p65) and non-classical (NIK-p100 / p52 / RelB) NF-κB pathways enhances the proliferation of malignant plasma cells and inhibits apoptosis. Among the extracellular signaling pathways activating NF-κB, CD40, a member of the TNF receptor superfamily, has become a key mediator. The binding of CD40 ligands (CD40L) expressed on bone marrow stromal and immune cells to CD40 on MM cells not only triggers NF-κB activation but also promotes environment-mediated drug resistance, enabling malignant cells to evade chemotherapy-induced cytotoxicity. Despite these findings, the spatial and functional regulation of CD40 / NF-κB signaling within MM cells, and which upstream regulators shape this axis, remain crucial biological and therapeutic questions.

[0004] In recent years, membrane surface receptors have become important regulators and therapeutic targets for multiple myeloma (MM). Among them, CD40, a member of the TNF receptor superfamily, has received increasing attention due to its central role in the pathobiology of MM. The binding of CD40 on MM cells to its ligand CD40L induces the production of large amounts of IL-6 by myeloma cells and stromal cells. This interaction promotes cell adhesion and migration and activates the NF-κB signaling pathway. Mechanistically, CD40 signaling co-activates both classical and non-classical NF-κB pathways, thereby enhancing the intrinsic NF-κB activity that drives MM cell survival, proliferation, and drug resistance. However, our understanding of the molecular regulators of the CD40 / NF-κB signaling axis remains incomplete. To fully elucidate and therapeutically target the aberrant activation of this pathway, it is crucial to identify and characterize novel regulators that influence its signaling dynamics. Emerging evidence suggests that malignant plasma cells not only rely on supportive signals from the bone marrow microenvironment but also require a cytoskeleton scaffold to maintain the spatial distribution and stability of membrane receptors, thereby sustaining the signaling cascade that promotes proliferation, survival, and drug resistance. For example, spectrin, actin, and their conjugate proteins (such as ankyrin and ERM family proteins ezrin, radixin, and moesin) have been shown to regulate immune synapse formation and receptor clustering, thereby modulating the activation of the NF-κB, MAPK, and PI3K-AKT pathways. Overexpression of ERM proteins bridges membrane receptors to the actin cytoskeleton, promotes the clustering of B cell receptors and adhesion molecules, and is associated with enhanced lymphocyte signaling, malignant transformation, migration, and drug resistance in hematologic malignancies.

[0005] Protein 4.1R (encoded by the EPB41 gene) is a core component of the membrane cytoskeleton, whose main functions include stabilizing the cytocortical cytoskeleton and tissue receptor complexes, and coupling them to downstream signaling pathways. The human EPB41 gene is located on the short arm of chromosome 1 at region 1p35.3, containing 21 exons and encoding four major functional domains—the FERM domain, the FERM adjacency domain (FA), the spectrin-actin binding domain (SAB), and the C-terminal domain (CTD). These domains collectively provide multiple interfaces for the interaction between membrane proteins and the cytoskeleton. Increasing evidence suggests that 4.1R also plays a signaling regulatory role in immune cells. In T cells, 4.1R is rapidly recruited to the immune synapse after TCR activation, acting as a potent negative regulator of early signaling events, effectively restraining T cell activation and proliferation. Conversely, in mast cells, 4.1R functions as a positive regulator of early activation triggered by FcεRI. In B cells, 4.1R co-localizes with membrane receptors such as TLR4 and selectively inhibits the classical NF-κB signaling pathway, thereby finely regulating the activation threshold and subsequent cell fate determination of B cells. These lineage-specific and often antagonistic roles suggest that 4.1R can both inhibit and enhance proximal receptor signaling, depending entirely on the specific cellular microenvironment and the receptor systems involved. Although the association between NF-κB overactivation and B-cell malignancies has been established, the pathogenic contribution and mechanistic role of 4.1R in B-cell-related cancers remain largely unknown. Summary of the Invention

[0006] The purpose of this invention is to provide the application of 4.1R protein as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, and the application of 4.1R protein in the preparation of drugs for the prevention and / or treatment of multiple myeloma.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: 4.1 Application of R protein as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma.

[0008] Furthermore, the 4.1R protein, as a scaffold protein of the CD40 / NF-κB signaling pathway, stabilizes CD40 localization and inhibits abnormal NF-κB activation by forming a complex with CD40 and NF-κB, thereby inhibiting the CD40 / NF-κB signaling pathway.

[0009] Furthermore, the 4.1R protein stabilizes CD40 localization by anchoring CD40 to the cell membrane.

[0010] Furthermore, the FERM and CTD domains of the 4.1R protein stabilize CD40 localization by anchoring CD40 to the cell membrane.

[0011] Furthermore, the FERM and CTD domains of the 4.1R protein stabilize CD40 localization by mediating the binding of the 4.1R protein to the cytoplasmic tail of CD40.

[0012] Furthermore, the NF-κB is NF-κB p50.

[0013] Furthermore, the FERM and CTD domains of the 4.1R protein reduce the nuclear translocation of NF-κBp50 by binding to it.

[0014] The 4.1R protein is used in the preparation of drugs for the prevention and / or treatment of multiple myeloma, wherein the 4.1R protein exerts an anti-cancer effect in multiple myeloma by inhibiting the CD40 / NF-κB signaling pathway, thereby inhibiting cell proliferation and cell cycle progression, promoting cell apoptosis.

[0015] Furthermore, the 4.1R protein exerts a tumor-suppressing effect in multiple myeloma by stabilizing CD40 localization and inhibiting abnormal NF-κB activation.

[0016] Furthermore, the FERM and CTD domains of the 4.1R protein exert a tumor-suppressive effect in multiple myeloma by binding to CD40 and NF-κB p50.

[0017] The beneficial effects of this invention are: This invention systematically investigated the functional mechanism of the 4.1R protein using flow cytometry analysis of bone marrow samples from multiple myeloma patients, bioinformatics methods, in vitro 4.1R knockdown and overexpression models, and an in vivo 5TGM1-Luc MM mouse model. Results showed that 4.1R protein expression gradually decreased with disease progression. In MM cells, 4.1R inhibited cell proliferation and cell cycle progression and promoted apoptosis by regulating CD40 / NF-κB signaling. Mechanistically, 4.1R anchors CD40 to the plasma membrane through its FERM and CTD domains, disrupting CD40 cluster formation and leading to abnormal NF-κB nuclear translocation. These findings indicate that 4.1R plays a tumor-suppressive role in MM by stabilizing CD40 localization, inhibiting NF-κB activation, and acting as a scaffold protein for the CD40 / NF-κB signaling axis. Attached Figure Description

[0018] Figure 1 The graph shows the correlation between downregulation of 4.1R protein expression in malignant plasma cells of multiple myeloma (MM) and disease progression, where A represents CD138 sorted from BMMCs of MM patients. ＋CD19 ＋ Plasma cell strategy diagram, B represents CD138 in CR and PD patients. ＋ CD19 ＋ Intracellular 4.1R expression level map, C represents CD138 in CR and PD patients. ＋ CD19 ＋ 4.1R in the group ＋ Quantitative analysis of cell proportions; *p<0.05; Figure 2 This diagram illustrates the regulation of apoptosis, cell cycle progression, and proliferation of MM cells by the 4.1R protein, where A represents the 4.1R protein detected by flow cytometry. low Group, normal control group and 4.1R high Image showing Annexin V / 7-AAD staining results of MM cells. B indicates PI staining cell cycle analysis showing 4.1R. low Group, normal control group and 4.1R high The distribution of cells in the G1, S, and G2 / M phases is shown in Figure C, which is the result of flow cytometry detection of EdU incorporation. Figure 3 For αCD40 stimulation, 4.1R low Group, normal control group and 4.1R high The effect of flow cytometry on apoptosis, cell cycle, and proliferation of MM cells is shown in Figure 4.1R. low Group, normal control group and 4.1R high Image showing Annexin V / 7-AAD staining results of MM cells. B indicates PI staining cell cycle analysis showing 4.1R. low Group, normal control group and 4.1R high The distribution of cells in the G1, S, and G2 / M phases is shown in Figure C, which is the result of flow cytometry detection of EdU incorporation. Figure 4 The images show the colocalization of the FERM and CTD domains of the 4.1R protein with CD40 and NF-κB p50 in MM cells. A is a confocal immunofluorescence image of MM cells; B shows the protein-protein interactions between 4.1R, CD40, and NF-κB p50; and C is the image of 4.1R... low Group, normal control group and 4.1R high Western blot analysis of nucleoplasmic components of MM cells, D = 4.1R low Immunofluorescence staining images of NF-κB p50 in MM cells of the control group and normal control group. E is a diagram of the potential spatial interaction between CD40, 4.1R membrane binding domains and NF-κB p50 predicted by the protein structure model. F is a diagram of the binding of 4.1R to CD40 mediated by HA-labeled FERM and CTD domains. Figure 5 The image shows an experiment designed to enhance tumor burden in a 5TGM1-induced MM mouse model by 4.1R protein deletion. A is a schematic diagram of the experimental design, and B is an immunohistochemical staining image of a decalcified femoral section with CD138. Detailed Implementation

[0019] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0020] Example 1 I. 4.1R expression is negatively correlated with the progression of multiple myeloma. Bone marrow mononuclear cells (BMMCs) from 5 patients with progressive disease (PD) and 5 patients with complete remission (CR) were analyzed by flow cytometry. BMMCs were isolated using a standardized protocol and analyzed using an established flow cytometry gating strategy. CD138 was also analyzed. + CD19 + Analysis of plasma cells showed that, from Figure 1 As shown in A and 1B, compared with CR cases, PD patients had a significantly reduced number of 4.1R-positive malignant plasma cells. This was achieved by quantifying CD138. + CD19 + 4.1R in plasma cell population + The proportion of plasma cells, from Figure 1 As can be seen from C, compared with CR patients, PD patients had a 4.1R + The proportion of plasma cells was significantly reduced. This negative correlation between 4.1R expression and disease activity supports the tumor-suppressive role of 4.1R in MM.

[0021] II. 4.1 R regulates apoptosis, cell cycle progression, and proliferation of multiple myeloma cells. A 4.1R low-expression variant was constructed using lentiviral transduction. low ) and 4.1R high expression (4.1R high The RPMI-8226 cell line was used, and a normal control group was established. From... Figure 2 Flow cytometry analysis showed that, compared with the normal control group and 4.1R high Compared to the previous group, 4.1R low The apoptosis rate of cells was significantly reduced (p<0.05). Cell cycle analysis showed that 4.1R low The proportion of cells in G1 phase decreased while the proportion in S phase increased, and 4.1R high The cells showed obvious G1 phase arrest and a decreased proportion of cells in S phase (p<0.05). Figure 2 C further confirms that 4.1R lowThe cells exhibited the highest DNA synthesis rate, while the normal control group was at a moderate level, 4.1R. high The lowest values ​​were observed in the group. These results collectively indicate that downregulation of 4.1R expression promotes proliferation and cell cycle progression while inhibiting apoptosis, while upregulation of 4.1R expression inhibits these processes, suggesting that it may be a therapeutic target for MM.

[0022] III. 4.1R as a scaffold-like signal aptamer bridging the CD40 and NF-κB pathways 4.1R low Normal control group, 4.1R high RPMI-8226 cells were stimulated with αCD40 agonist at a concentration of 2 µg / mL for 24 hours, followed by flow cytometry analysis, and compared with the PBS (Control) group. Figure 3 Apoptosis experiments showed that αCD40 stimulation significantly reduced Annexin V in the normal group. + Cells, and 4.1R low No significant changes were observed in the expression group, 4.1R. high Only a slight and insignificant reduction was observed in the expression group. Figure 3 Cell cycle data from B cells showed that αCD40 stimulation promoted normal cell cycle activity and 4.1R cell cycle activity. high The expression cells entered the S phase and reduced the number of cells in the G1 phase. Figure 3 C further confirmed that αCD40-induced proliferation occurred in normal and 4.1R... high Expression was significantly increased in cells, but at 4.1R low Expression was not increased in cells. These findings collectively demonstrate that 4.1R is a key signaling relay station in the CD40 pathway, transmitting proliferation and anti-apoptotic signals in MM cells.

[0023] Immunofluorescence technology utilizes the specific binding of fluorescently labeled antibodies to antigens in cells and tissues, allowing observation of antigen localization and distribution under a fluorescence microscope. Its core is the antigen-antibody reaction, using fluorescein-labeled antibodies as probes; upon excitation light, these antibodies emit fluorescent signals for visualization. Immunofluorescence experiments show that 4.1R (red) and CD40 (purple) are mainly distributed on the cell membrane, and fluorescence intensity analysis reveals significant co-localization between the two on the membrane (r=0.9472, p<0.0001). Figure 4A) indicates a strong interaction between 4.1R and CD40. Co-immunoprecipitation (Co-IP) is a technique based on antigen-antibody specific binding used to study protein interactions under physiological conditions. Its core principle is to capture the target protein with an antibody and precipitate the interacting protein that it naturally binds to. Co-immunoprecipitation experiments using anti-4.1R and anti-CD40 antibodies showed an interaction between 4.1R and CD40 in MM cell lines (RPMI-8226 and MM.1S), confirming the direct binding of 4.1R to CD40. Figure 4 B). From Figure 4 Western blot analysis of C showed that silencing 4.1R significantly enhanced the accumulation of NF-κB p50 in the cell nucleus. This result was obtained through... Figure 4 D further confirmed that, compared with the normal group, the localization of NF-κB p50 in the nucleus was significantly increased in cells with low expression of 4.1R. Figure 4 In C, GAPDH is an internal reference.

[0024] Computer modeling using Chimera software revealed that the membrane-binding FERM domain of 4.1R can simultaneously interact with CD40 and NF-κB p50, providing mechanistic support for the scaffold function of 4.1R. Domain localization experiments were conducted in 293T cells using FLAG-labeled CD40 constructs and HA-labeled full-length and truncated 4.1R constructs. The results showed that the FERM and CTD domains of 4.1R can directly bind to CD40. This result validates the predictions made by Chimera software and reveals a key mechanism by which 4.1R regulates CD40.

[0025] These results indicate that the 4.1R protein acts as a scaffold adapter in the CD40 / NF-κB signaling pathway. By anchoring CD40 to the cell membrane and inhibiting aberrant NF-κB activation, 4.1R maintains a balanced signal output regulating MM cell survival, proliferation, and disease progression. Knockdown of 4.1R disrupts this regulatory platform, leading to enhanced NF-κB nuclear translocation and a feedback upregulation of CD40 expression, thereby driving a malignant signaling cascade.

[0026] IV. 4.1R Defects Exacerbate Tumor Burden in 5TGM1-Induced Myeloma Models For 6-8 week old C57BL / 6J 4.1R ⁺ / ⁺ and 4.1R - / - Mice were anesthetized and injected intratibially with 1×10 6Five TGM1-Luciferase cells were suspended in 50 μL of PBS. Engraftment and early tumor growth were assessed by bioluminescence imaging (BLI) on day 14 after D-fluorescein administration, and quantified by total photon flux (photons / second). Mice were sacrificed on day 21, and femurs were harvested for decalcification, paraffin embedding, and CD138 immunohistochemistry to assess bone marrow plasma cell infiltration. This method enabled MM cells to engraft in the bone marrow microenvironment, closely mimicking the clinical environment of human MM. Tumor progression was monitored longitudinally by BLI on days 14 and 21 after intraperitoneal injection of D-fluorescein potassium, and tumor burden was further assessed by immunohistochemical analysis of femoral sections. BLI results showed that, compared with 4.1R... + / + Compared with the control group, 4.1R - / - The tumor signal was stronger in mice, and further histological analysis showed that 4.1R - / - CD138 in mouse femoral bone marrow + Plasma cell infiltration was significantly enhanced. In conclusion, 4.1R exerts an antitumor effect in vivo, inhibiting the growth and infiltration of MM cells in the bone marrow microenvironment.

[0027] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. 4.1 Application of R protein as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma.

2. The application of the 4.1R protein according to claim 1 as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, characterized in that, The 4.1R protein, as a scaffold protein of the CD40 / NF-κB signaling pathway, stabilizes CD40 localization and inhibits abnormal NF-κB activation by forming a complex with CD40 and NF-κB, thereby inhibiting the CD40 / NF-κB signaling pathway.

3. The application of the 4.1R protein according to claim 2 as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, characterized in that, The 4.1R protein stabilizes CD40 localization by anchoring CD40 to the cell membrane.

4. The application of the 4.1R protein according to claim 2 or 3 as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, characterized in that, The FERM and CTD domains of the 4.1R protein stabilize CD40 localization by anchoring it to the cell membrane.

5. The application of the 4.1R protein according to claim 2 or 3 as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, characterized in that, The FERM and CTD domains of the 4.1R protein stabilize CD40 localization by mediating the binding of the 4.1R protein to the cytoplasmic tail of CD40.

6. The application of the 4.1R protein according to claim 2 as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, characterized in that, The NF-κB mentioned is NF-κB p50.

7. The application of the 4.1R protein according to claim 2 or 6 as an inhibitor of the CD40 / NF-κB signaling pathway in multiple myeloma, characterized in that, The FERM and CTD domains of the 4.1R protein reduce the nuclear translocation of NF-κB p50 by binding to it. 8.4.1 The use of R protein in the preparation of drugs for the prevention and / or treatment of multiple myeloma, characterized in that, The 4.1R protein exerts an anti-cancer effect in multiple myeloma by inhibiting the CD40 / NF-κB signaling pathway, thereby suppressing cell proliferation and cell cycle progression, and promoting apoptosis.

9. The application of the 4.1R protein according to claim 8 in the inhibition of multiple myeloma drugs, characterized in that, The 4.1R protein exerts a tumor-suppressive effect in multiple myeloma by stabilizing CD40 localization and inhibiting abnormal NF-κB activation.

10. The application of the 4.1R protein according to claim 8 in the inhibition of multiple myeloma drugs, characterized in that, The FERM and CTD domains of the 4.1R protein exert a tumor-suppressive effect in multiple myeloma by binding to CD40 and NF-κB p50.

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