A molecular mechanism regulating the stability of FGFR1 protein and its application

Abstract

This invention discloses a molecular mechanism for regulating FGFR1 protein stability and its applications. For the first time, this invention reveals that OLFM4, as an FGFR1 binding protein, stabilizes the FGFR1 protein and maintains the continuous activation of its downstream MAPK-AP1 signaling pathway by antagonizing the FGFR1 ubiquitination degradation mediated by the E3 ubiquitin ligase TRIM21. Through experiments including bimolecular fluorescence complementation, immunoprecipitation, truncated mutant analysis, protein stability tracking, ubiquitination detection, and clinical sample validation, this invention systematically elucidates the molecular basis of the OLFM4-TRIM21-FGFR1 regulatory axis. Based on this mechanism, this invention provides a method for screening drugs that regulate FGFR1 stability, and the application of OLFM4 inhibitors in the preparation of drugs for treating diseases related to abnormal activation of the FGFR signaling pathway (especially biliary tract tumors). This invention provides a novel non-ATP-competitive intervention target for therapeutic strategies targeting the FGFR1 signaling pathway, possessing significant scientific value and promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to a molecular mechanism by which the stability of FGFR1 protein is regulated through the OLFM4-TRIM21-FGFR1 ternary complex, and the application of this mechanism in drug screening, preparation of drug compositions, and treatment of diseases related to abnormal activation of the FGFR signaling pathway. Background Technology

[0002] Fibroblast growth factor receptor 1 (FGFR1) is an important member of the receptor tyrosine kinase family, playing a crucial role in biological processes such as cell proliferation, differentiation, migration, and survival. Aberrant activation of FGFR1, whether through gene amplification, mutation, or overexpression, has been shown to effectively drive downstream signaling pathways such as RAS-MAPK, promoting tumor proliferation, invasion, and drug resistance. In various malignant tumors, including lung cancer, breast cancer, glioblastoma, and biliary tract tumors, high expression or aberrant activation of FGFR1 is closely associated with poor patient prognosis.

[0003] Protein stability regulation is one of the core mechanisms for maintaining cellular signal homeostasis, with the ubiquitin-proteasome system (UPS) being the most important protein degradation pathway. E3 ubiquitin ligases recognize substrate proteins and catalyze the covalent attachment of ubiquitin molecules to the substrate, labeling the substrate protein for recognition and degradation by the 26S proteasome. Trim21 (TRIM21) is an important member of the TRIM family of E3 ubiquitin ligases, its structure including an N-terminal RING domain (possessing E3 ubiquitin ligase activity), a B-box domain, a coiled-coil domain, and a C-terminal PRY / SPRY domain. Studies have shown that TRIM21 participates in various pathological processes such as inflammation, autoimmunity, and cancer, exerting its biological functions by ubiquitinizing and degrading various substrate proteins, such as the apoptosis inhibitor c-FLIP, BCL2, and the transcription factor SALL4. However, whether TRIM21 regulates the protein stability of FGFR1, and how it is regulated, remains unreported.

[0004] Oligosin 4 (OLFM4), a member of the olfactory protein family, was initially identified in human myeloid leukemia cells and subsequently found to be expressed in gastric mucosal epithelium, intestinal crypt basal cells, and some myeloid cells. In oncology, the role of OLFM4 exhibits a high tumor type dependence. Our previous research found that OLFM4 mediates tumor cell immune escape in gallbladder cancer by activating the MAPK-AP1 signaling axis and upregulating PD-L1 expression. However, whether OLFM4 regulates upstream signaling pathways by affecting protein stability, particularly its molecular associations with FGFR1 and TRIM21, remains unclear.

[0005] Therefore, elucidating the regulatory mechanisms of FGFR1 protein stability and identifying key modifiable molecular nodes is of significant scientific and clinical value for developing therapeutic strategies targeting the FGFR1 signaling pathway. This invention, based on a systematic study of the OLFM4-TRIM21-FGFR1 regulatory axis, reveals for the first time that OLFM4, as a stable protein of FGFR1, maintains FGFR1 protein stability and continuously activates downstream signaling pathways by antagonizing TRIM21-mediated FGFR1 ubiquitination and degradation, providing a novel intervention target and strategy for treating FGFR-related diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular mechanism for regulating the stability of FGFR1 protein, specifically involving the interaction and functional regulation among OLFM4, TRIM21 and FGFR1.

[0007] Another objective of this invention is to provide a drug screening method and its application based on this molecular mechanism.

[0008] Another object of the present invention is to provide the application of OLFM4 inhibitors in the preparation of drugs for treating diseases related to abnormal activation of the FGFR signaling pathway.

[0009] 1. Elucidation of the molecular mechanism This invention reveals for the first time the existence of the OLFM4-TRIM21-FGFR1 regulatory axis and its central role in regulating FGFR1 protein stability. Through a series of molecular biology experiments, this invention confirms that: (1) OLFM4 and FGFR1 have a direct physical interaction. The binding between the two was verified at the live cell level by bimolecular fluorescence complementation (BiFC) assay; the specificity of this interaction was further confirmed by exogenous immunoprecipitation (Co-IP) assay, and it was found that co-precipitation could still occur between human FGFR1 and rat OLFM4, suggesting that this interaction is conserved across species. By constructing a series of truncated mutants of FGFR1, it was found that immunoglobulin-like domains I and III of FGFR1 are the regions necessary for its binding with OLFM4, while the deletion of domain II enhanced the binding, suggesting that it may play a negative regulatory role.

[0010] (2) OLFM4 promotes the accumulation of FGFR1 in the cytoplasm and prolongs its half-life. Subcellular component separation experiments showed that FGFR1 is mainly located in the cytoplasm, and OLFM4 treatment increased the amount of FGFR1 protein in the cytoplasm in a time-dependent manner. Immunofluorescence experiments further confirmed that compared with FGF2 treatment, OLFM4 treatment resulted in more significant fluorescent accumulation of FGFR1 in the cytoplasm. Actinomycin tracing experiments showed that under conditions of inhibited transcription and protein synthesis, OLFM4 significantly slowed down the degradation rate of FGFR1 protein and maintained the phosphorylation levels of downstream p-FGFR1, p-MEK1 / 2, and p-ERK1 / 2. FGF2 combined with OLFM4 overexpression experiments showed that the addition of OLFM4 enhanced the amount of FGFR1 protein and its phosphorylation level, and upregulated the expression of downstream AP-1 family members such as c-JUN and ATF3.

[0011] (3) Interaction between OLFM4 and the E3 ubiquitin ligase TRIM21. Using LC-MS / MS mass spectrometry combined with immunoprecipitation to screen for interacting proteins of OLFM4, TRIM21 was identified as a candidate interacting protein. Two-way immunoprecipitation experiments confirmed that TRIM21 not only interacts with OLFM4 but also co-precipitates with FGFR1, suggesting that TRIM21 may act as a bridging molecule involved in the regulation of the OLFM4-FGFR1 complex.

[0012] (4) TRIM21 mediates the ubiquitination and degradation of FGFR1, while OLFM4 inhibits this process. Proteasome / lysosome inhibition assays showed that TRIM21 mainly relies on the proteasome pathway for degradation. Ubiquitination assays confirmed that TRIM21 overexpression significantly promotes the polyubiquitination modification of FGFR1; while co-expression of OLFM4 and TRIM21 effectively inhibits TRIM21-mediated FGFR1 ubiquitination, thereby stabilizing the FGFR1 protein.

[0013] (5) Validation of clinical tissue samples. Immunohistochemical staining showed that in biliary tract tumor tissues with high OLFM4 expression, FGFR1, TRIM21, p-ERK1 / 2, CCL2, and the neutrophil marker CD66b all showed significant positive staining, and the positive signals were mainly located in the cytoplasm of tumor cells and the infiltrated immune cell regions; while in the OLFM4 low expression group, the staining intensity of the above targets was generally weakened. Survival analysis based on molecular axis activity scores showed that the overall survival of patients in the high-score group was significantly lower than that in the low-score group, suggesting that the activation of this signal axis is closely related to poor patient prognosis.

[0014] Based on the above findings, this invention proposes a working model of the "OLFM4-TRIM21-FGFR1" regulatory axis: When OLFM4 expression is upregulated, OLFM4 forms a complex with TRIM21, altering the catalytic activity or spatial orientation of TRIM21 and inhibiting its ubiquitination modification of FGFR1, thereby stabilizing the FGFR1 protein and prolonging its signal output time; the stabilized FGFR1 further activates the downstream MAPK-AP1 signaling cascade, upregulates the expression of chemokines such as CCL2, promotes the recruitment of immature neutrophils and the formation of NETosis, reshapes the immunosuppressive microenvironment, and drives the malignant progression of tumors.

[0015] 2. Drug screening methods Based on the above molecular mechanism, this invention provides a method for screening candidate substances that regulate the stability of FGFR1 protein, comprising the following steps: a) Contact the analyte with a system containing OLFM4, TRIM21 and FGFR1; b) Detect the ubiquitination level or stability of FGFR1 protein in the system; and c) Compared with the control, if the test substance can promote the binding of OLFM4 to TRIM21, or inhibit the binding of TRIM21 to FGFR1, or directly reduce the ubiquitination level of FGFR1, then the test substance is identified as a candidate substance for regulating the stability of FGFR1 protein.

[0016] The detection methods described in step b) include, but are not limited to, co-precipitation immunoassay, Western blotting, immunofluorescence, or bimolecular fluorescence complementation assay.

[0017] 3. Application This invention provides the use of OLFM4 protein or a functional fragment thereof in the preparation of drugs or kits for stabilizing FGFR1 protein.

[0018] This invention provides the use of TRIM21 protein or a functional fragment thereof in the preparation of drugs or kits for promoting the degradation of FGFR1 protein.

[0019] This invention provides the use of an inhibitor of the OLFM4 protein in the preparation of a medicament for treating diseases associated with abnormal activation of the FGFR1 signaling pathway. The inhibitor of the OLFM4 protein, by blocking the binding of OLFM4 to TRIM21, relieves the inhibitory effect of OLFM4 on TRIM21-mediated FGFR1 ubiquitination and degradation. The diseases associated with abnormal activation of the FGFR1 signaling pathway are preferably biliary tract tumors, particularly intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, or gallbladder cancer.

[0020] The inhibitor of the OLFM4 protein may be selected from one or more of the following groups: siRNA, shRNA, antisense oligonucleotides, CRISPR gene editing systems, specific antibodies or their antigen-binding fragments, and small molecule compounds that target OLFM4.

[0021] The present invention also provides the application of biomaterials containing OLFM4, TRIM21 and FGFR1 in constructing a model for screening drugs that regulate the stability of FGFR1 protein.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to discover and confirm that OLFM4, as a binding protein of FGFR1, stabilizes the FGFR1 protein and maintains the continuous activation of downstream signals by competitively inhibiting TRIM21-mediated FGFR1 ubiquitination and degradation. This discovery fills the gap in understanding how OLFM4, as a secretory protein, initiates intracellular signal transduction and provides a new perspective on the regulation of post-translational modifications of FGFR1.

[0023] 2. The assembly interface of the OLFM4-TRIM21-FGFR1 ternary complex disclosed in this invention, especially the immunoglobulin-like domains I and III of FGFR1, can serve as novel intervention targets for small molecule drugs, peptides, or biologics, providing the possibility for developing non-ATP-competitive FGFR signaling pathway inhibitors.

[0024] 3. Based on the core role of OLFM4 in stabilizing FGFR1, inhibitors targeting OLFM4 (such as neutralizing antibodies and nucleic acid drugs) can effectively block the FGFR1 signaling pathway, providing a new treatment strategy for FGFR-related tumors. In particular, combined use with immune checkpoint inhibitors may produce synergistic anti-tumor effects.

[0025] 4. This invention, through clinical tissue sample validation and survival analysis, confirms that the activity of the OLFM4-driven FGFR1-CCL2 signal axis is closely related to the poor prognosis of patients with biliary tract tumors, suggesting that this signal axis can serve as a biomarker for judging patient prognosis and provide a basis for precision clinical treatment. Attached Figure Description

[0026] Figure 1 BiFC verification experiment of OLFM4 and FGFR1 interaction; (A) Bimolecular fluorescence complementation (BiFC) experiment showed that after co-transfection of HEK293T cells with OLFM4-VN173 and FGFR1-VC155, bright green fluorescence was visible under confocal microscopy, indicating that the two have a direct interaction in living cells; (B) positive control verification of BiFC experiment.

[0027] Figure 2 Immunoprecipitation of OLFM4-FGFR1 interaction and FGFR1 domain elucidation; (AB) exogenous immunoprecipitation verifies the interaction between OLFM4 and FGFR1. Repeated verification using different tag combinations (Flag / HA, His / HA) confirms the specificity of their interaction; (C) FGFR1 domain elucidation. By constructing a series of truncated FGFR1 mutants (△1, △2, △3), it was found that immunoglobulin-like domains I and III are essential for binding to OLFM4, while domain II may play a negative regulatory role.

[0028] Figure 3 Subcellular localization and protein stability regulation of FGFR1 by OLFM4; (A) Subcellular component separation experiment in GBC-SD cells showed that FGFR1 was mainly located in the cytoplasm, and the amount of FGFR1 protein in the cytoplasm increased in a time-dependent manner after OLFM4 treatment; (B) Actinomycin tracing experiment showed that under the condition of inhibited transcription and protein synthesis, OLFM4 significantly slowed down the degradation rate of FGFR1 protein and maintained the phosphorylation levels of downstream p-FGFR1, p-MEK1 / 2, and p-ERK1 / 2.

[0029] Figure 4 Verification of OLFM4's effect on FGFR1 protein stability and downstream signaling pathways; (A) Immunofluorescence analysis showed that compared with FGF2 treatment, OLFM4 treatment resulted in more significant fluorescence accumulation of FGFR1 in the cytoplasm; (B) Protein level verification showed that FGF2 combined with OLFM4 overexpression could enhance the amount of FGFR1 protein and its phosphorylation level, and upregulate the expression of downstream AP-1 family members such as c-JUN and ATF3.

[0030] Figure 5 LC-MS / MS identification of OLFM4 interacting proteins and verification of TRIM21 interaction; (A) LC-MS / MS combined with Flag-IP to identify OLFM4 interacting proteins, Venn plot showed that 51 unique proteins were identified in the Flag-OLFM4 group, including TRIM21; (B) Immunoprecipitation to verify the interaction between OLFM4 and TRIM21; (C) Immunoprecipitation to verify the interaction between FGFR1 and TRIM21.

[0031] Figure 6OLFM4 regulates TRIM21-mediated FGFR1 ubiquitination and degradation; (A) Inhibitor screening experiments of protein degradation pathway showed that MG132 treatment significantly enhanced TRIM21 protein levels, suggesting that TRIM21 mainly depends on the proteasome pathway for degradation; (B) Ubiquitination detection experiments showed that TRIM21 overexpression significantly promoted FGFR1 polyubiquitination modification, while OLFM4 co-expression with TRIM21 effectively inhibited this modification, thereby stabilizing FGFR1 protein.

[0032] Figure 7 Immunohistochemical staining was used to detect the association between the OLFM4-mediated FGFR1-CCL2 signaling axis and the tumor immune microenvironment in clinical tissue samples. In the OLFM4 high expression group, FGFR1, TRIM21, p-ERK1 / 2, CCL2 and the neutrophil marker CD66b all showed significant positive staining. In the OLFM4 low expression group, the staining intensity of the above targets was generally weakened.

[0033] Figure 8 Survival analysis of OLFM4-driven FGFR1-CCL2 signal axis activity and prognosis in patients with biliary tract tumors; Kaplan-Meier survival curves based on molecular axis activity score (GSVA score) showed that the overall survival of patients in the high-score group was significantly lower than that in the low-score group (p=0.018), suggesting that activation of this signal axis is closely related to poor patient prognosis. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Example 1: Verification of the interaction between OLFM4 and FGFR1 1. Experimental Objective This embodiment aims to verify whether there is a direct physical interaction between OLFM4 and FGFR1, and to determine the subcellular localization of their interaction.

[0036] 2. Experimental Materials (1) Cells: Human embryonic kidney HEK293T cells and human gallbladder cancer cells GBC-SD were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0037] (2) Main reagents: DMEM medium, fetal bovine serum, penicillin-streptomycin, liposome transfection reagent, anti-quenching mounting medium, DAPI, 4% paraformaldehyde, RIPA lysis buffer, protease inhibitor mixture, protein A / G magnetic beads.

[0038] (3) Major antibodies: anti-Flag, anti-HA, anti-His, anti-FGFR1, anti-GAPDH, Alexa Fluor 488-labeled goat anti-rabbit IgG, Alexa Fluor 594-labeled goat anti-rabbit IgG.

[0039] (4) Major plasmids: pBiFC-VN173-OLFM4, pBiFC-VC155-FGFR1, pcDNA3.1-Flag-OLFM4, pcDNA3.1-HA-FGFR1, pcDNA3.1-His-rOLFM4 (rat recombinant OLFM4).

[0040] (5) Main instruments: Leica TCS SP8 confocal microscope, CO2 constant temperature incubator, ultra-clean workbench, low temperature high speed centrifuge.

[0041] 3. Experimental Methods 3.1 Bimolecular fluorescence complementation experiment The OLFM4 coding sequence was cloned into the pBiFC-VN173 vector to construct the OLFM4-VN173 fusion expression plasmid; the FGFR1 coding sequence was cloned into the pBiFC-VC155 vector to construct the FGFR1-VC155 fusion expression plasmid. These plasmids were co-transfected into HEK293T cells using liposome transfection. Alternative negative controls included transfection with OLFM4-VN173, FGFR1-VC155, and co-transfection with the empty vector. Forty-eight hours after transfection, the culture medium was discarded, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and then stained with DAPI for nuclear staining. Images were acquired using a Leica TCS SP8 confocal microscope with an excitation wavelength of 488 nm, and green fluorescence signals were detected.

[0042] 3.2 Exogenous Immunoprecipitation Assay Exogenous immunoprecipitation was validated in HEK293T cells. Two groups were set up for the experiment: (1) HEK293T cells were co-transfected with OLFM4 carrying the Flag tag (OLFM4-Flag) and FGFR1 carrying the HA tag (FGFR1-HA); (2) HEK293T cells were co-transfected with FGFR1 carrying the HA tag (FGFR1-HA) and rat recombinant OLFM4 carrying the His tag (His-rOLFM4). 48 hours after transfection, cells were collected, and IP lysis buffer containing a mixture of protease inhibitors was added. Cells were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. A portion of the supernatant was used as input. 2 μg of the corresponding tag antibody (anti-Flag, anti-HA, or anti-His) was added to the remaining supernatant, and the cells were incubated overnight at 4°C. Protein A / G magnetic beads were added the next day and incubated at 4°C for 4 hours. The magnetic beads were collected using a magnetic rack and washed 5 times with IP lysis buffer. Add 2× loading buffer, denature at 95℃ for 10 minutes, collect the eluent for Western blotting, and perform immunoblotting using the corresponding tag antibodies.

[0043] 3.3 Subcellular component separation experiment Subcellular component separation kits were used to separate cytoplasmic and nuclear proteins from GBC-SD cells. Cells were collected, washed twice with PBS, and incubated with cytoplasmic extraction buffer for 15 minutes on ice. The cells were then centrifuged at 1000g for 5 minutes at 4°C, and the supernatant was collected as the cytoplasmic protein fraction. The precipitate was then incubated with nuclear extraction buffer for 30 minutes on ice, and centrifuged at 15000g for 10 minutes at 4°C, and the supernatant was collected as the nuclear protein fraction. The protein concentration of each fraction was determined using the BCA method. Equal amounts of protein were analyzed by Western blotting: FGFR1 protein levels were detected with FGFR1 antibody; nuclear protein purity was verified with ATF3 antibody; the specificity of the nuclear fraction was further confirmed with LaminB antibody; and the specificity of the cytoplasmic fraction was confirmed with GAPDH antibody. Subcellular distribution and protein level changes of FGFR1 were observed at different time points (0h, 3h, 6h).

[0044] 3.4 Immunofluorescence staining and colocalization analysis Immunofluorescence staining was performed in GBC-SD cells. Cells were cultured to 70% confluence and treated with either FGF2 (50 ng / ml) or recombinant OLFM4 protein (100 ng / ml) at 0 min, 30 min, 3 h, and 6 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 10% normal goat serum for 30 min. Rabbit anti-human FGFR1 primary antibody (1:200) was added and incubated overnight at 4°C. The next day, after washing, goat anti-rabbit IgG labeled with Alexa Fluor 594 (1:500) was added and incubated at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI and mounted with anti-quenching mounting medium. Images were acquired using a Leica TCS SP8 confocal microscope to compare the intracellular localization changes and fluorescence intensity differences of FGFR1 in different treatment groups and at different time points.

[0045] 4. Experimental Results The results of the bimolecular fluorescence complementation assay showed that after co-transfection of HEK293T cells with OLFM4-VN173 and FGFR1-VC155, the cells exhibited bright green fluorescence under a confocal microscope, while the negative control group, which was transfected alone or with an empty vector, showed no fluorescence signal. This indicates that there is indeed a direct physical interaction between OLFM4 and FGFR1 in living cells. Figure 1 ).

[0046] Exogenous immunoprecipitation assays showed that in the OLFM4-Flag+FGFR1-HA co-transfected group, a HA-FGFR1-specific band was detected by Western blotting after immunoprecipitation with anti-Flag antibody; conversely, a Flag-OLFM4-specific band was detected after immunoprecipitation with anti-HA antibody. In the FGFR1-HA+His-rOLFM4 co-transfected group, a His-rOLFM4 positive signal was detected after immunoprecipitation with anti-HA antibody; similarly, a HA-FGFR1 signal was detected after immunoprecipitation with anti-His antibody. These results indicate that the interaction between Flag-OLFM4 and HA-FGFR1 is not affected by the tag type, and that a protein complex can still be formed between human FGFR1 and rat OLFM4, suggesting that the binding of OLFM4 to FGFR1 is tag-independent and cross-species conserved. Figure 2 AB).

[0047] Subcellular component separation experiments showed that the FGFR1 protein level in the cytoplasm gradually increased from the baseline level at 0 h, and was significantly higher at 6 h than at 0 h; in the nuclear group, the FGFR1 protein level remained relatively stable, maintaining a level close to the baseline level at 0 h, without any significant increase or decrease. Figure 3 A). This indicates that the main functional regulation of FGFR1 occurs in the cytoplasm.

[0048] Immunofluorescence assays showed that in the FGF2 treatment group, FGFR1 was mainly distributed in the cytoplasm at 0 min; the fluorescence intensity of FGFR1 in the cytoplasm increased slightly at 30 min; the fluorescence intensity of FGFR1 in the cytoplasm and near the cell membrane significantly increased at 3 h; and the fluorescence continued to increase at 6 h. In the OLFM4 treatment group, FGFR1 was mainly located in the cytoplasm at 0 min; the fluorescence intensity of FGFR1 in the cytoplasm significantly increased at 3 h; and the fluorescence intensity further increased at 6 h. Compared with the FGF2 group at the same time point, the fluorescence accumulation of FGFR1 in the cytoplasm was more significant after OLFM4 treatment. Figure 4 A). The above results indicate that OLFM4 can promote the accumulation of FGFR1 in the cytoplasm.

[0049] Example 2: Identification of the FGFR1-OLFM4 binding domain 1. Experimental Objective This embodiment aims to identify the specific region on the FGFR1 protein responsible for binding to OLFM4.

[0050] 2. Experimental Materials Same as Example 1.

[0051] 3. Experimental Methods Based on the domain characteristics of the FGFR1 protein, a series of truncated mutants of FGFR1 were constructed: FGFR1-△1 (deficient in immunoglobulin-like domain I), FGFR1-△2 (deficient in immunoglobulin-like domain II), and FGFR1-△3 (deficient in immunoglobulin-like domain III). Meanwhile, full-length FGFR1 with the f1 origin of replication (FGFR1-f1) was used as a positive control, and an empty vector was used as a negative control. OLFM4-Flag and the aforementioned FGFR1 mutants were co-transfected into HEK293T cells, and cell lysates were collected 48 hours after transfection. OLFM4-Flag was enriched by immunoprecipitation using an anti-Flag antibody, and the complex was eluted and analyzed by Western blotting. The co-precipitated FGFR1 and its mutants were detected using an anti-HA antibody, and the binding ability of each mutant to OLFM4 was compared, with the intensity of the co-precipitated band of full-length FGFR1-f1 used as the reference standard.

[0052] 4. Experimental Results Immunoprecipitation results showed that full-length FGFR1-f1 could be effectively coprecipitated in the presence of OLFM4-Flag, while no nonspecific bands were observed in the empty vector group, confirming the specificity of the experimental system. Specifically, the binding affinity of FGFR1-Δ2 to OLFM4 was significantly enhanced, while the coprecipitation signals of FGFR1-Δ1 and FGFR1-Δ3 with OLFM4 were significantly weakened, with band intensities much lower than those of full-length FGFR1-f1. Figure 2 C). This indicates that the domains corresponding to △1 and △3 are crucial for maintaining the stable binding of FGFR1 and OLFM4, and are necessary regions for binding; while the domain corresponding to △2 may play a negative regulatory role or is not necessary for binding, and its absence may actually enhance the affinity with OLFM4.

[0053] Example 3: Regulation of FGFR1 protein stability by OLFM4 1. Experimental Objective This embodiment aims to investigate the regulatory role of OLFM4 on the stability of FGFR1 protein and its impact on downstream signaling pathways.

[0054] 2. Experimental Materials Same as Example 1. Also required are actinomycin D (CHX), recombinant human FGF2 protein, and cycloheximide (CHX).

[0055] 3. Experimental Methods 3.1 Actinomycete ketone tracking experiment A cyclohexane tracking assay was conducted in GBC-SD cells. Experimental groups included: control group (no treatment), OLFM4 treatment group (100 ng / ml), CHX group (50 μg / ml protein synthesis inhibitor), CHX+OLFM4 group, Acti D group (5 μg / ml transcription inhibitor), and Acti D+OLFM4 group. Treatment time points were 0 h, 3 h, and 6 h. Cells were collected at each time point, and total protein was extracted for Western blotting. The assay parameters included FGFR1, p-FGFR1, MEK1 / 2, p-MEK1 / 2, ERK1 / 2, and p-ERK1 / 2. Using GAPDH as an internal control, the effect of OLFM4 on the degradation rate of FGFR1 was analyzed by comparing the protein band intensities at each time point.

[0056] 3.2 Combined experiment of ligand stimulation and OLFM4 overexpression A combined treatment experiment was conducted in GBC-SD cells involving stimulation with recombinant FGF2 and overexpression of OLFM4. Experimental groups included: control group, rFGF2 group (50 ng / ml), rFGF2+OLFM4 overexpression group, and OLFM4 overexpression group. Treatment time points were 0 h, 1 h, 3 h, 6 h, and 12 h. Total cellular protein was collected at each time point and analyzed by Western blotting. The detected indicators included: FGFR1, p-FGFR1, MEK1 / 2, p-MEK1 / 2, ERK1 / 2, p-ERK1 / 2, c-JUN, and ATF3.

[0057] 4. Experimental Results The results of the actinomycin D tracking experiment showed that, under the background of co-treatment with CHX and Actinomycin D or treatment alone, the degradation rate of FGFR1 protein was significantly slowed at 3h and 6h after the addition of OLFM4 compared with the group without OLFM4. The protein levels of downstream signaling molecules p-FGFR1, p-MEK1 / 2, and p-ERK1 / 2 in the OLFM4-treated group showed a trend consistent with the changes in FGFR1 protein stability; the total protein expression of MEK1 / 2 and ERK1 / 2 was less affected by protein degradation and transcriptional repression in the OLFM4-treated group. Figure 3 B). This indicates that under conditions of inhibited transcription and protein synthesis, OLFM4 can slow down the degradation rate of FGFR1 protein, suggesting that OLFM4 may stabilize the protein by inhibiting the ubiquitination and degradation of FGFR1.

[0058] The combined ligand stimulation and OLFM4 overexpression experiments showed that under rFGF2 stimulation, the protein level of FGFR1 increased over time, while the addition of rOLFM4 further enhanced FGFR1 protein expression; compared with the group without rOLFM4, the band intensity of p-FGFR1 in the group with rOLFM4 was enhanced; the band intensity of p-MEK1 / 2 and p-ERK1 / 2 was enhanced under rFGF2 stimulation; the protein level of c-JUN increased over time under rFGF2 stimulation, while the protein level of ATF3 showed a trend of first increasing and then decreasing. Figure 4 B). The above results indicate that OLFM4 is involved in regulating the protein stability of FGFR1, affecting the activation state of FGFR1, and further enhancing the downstream MAPK-AP-1 pathway.

[0059] Example 4: Mass spectrometry identification of OLFM4 interacting proteins and verification of TRIM21 1. Experimental Objective This embodiment aims to systematically identify the interacting proteins of OLFM4 and verify the interaction between TRIM21 and OLFM4 and FGFR1.

[0060] 2. Experimental Materials Same as Example 1. Also required: Q Exactive mass spectrometer and MaxQuant software.

[0061] 3. Experimental Methods 3.1 Identification by co-immunoprecipitation combined with mass spectrometry HEK293T cells were transfected with Flag-OLFM4 and an empty vector (negative control), with three biological replicates for each group. Cell lysates were collected 48 hours after transfection and subjected to immunoprecipitation using an anti-Flag affinity gel. After eluting the complex, the cells were separated by SDS-PAGE electrophoresis. Following Coomassie brilliant blue staining, each lane was divided into 10 gel strips for in-gel digestion. The digested peptides were desalted using a C18 column and analyzed by LC-MS / MS using a Q Exactive mass spectrometer. Raw data were analyzed using MaxQuant software for library search and identification, with a false positive rate of <1%. Protein identification results were compared between the IgG and Flag-OLFM4 groups, and proteins specific to the Flag-OLFM4 group with a peptide count ≥2 were selected as candidate interacting proteins.

[0062] 3.2 TRIM21 Interaction Verification Based on mass spectrometry identification results, the interaction between TRIM21 and OLFM4 and FGFR1 was verified by immunoprecipitation. Experiment 1: HEK293T cells were transfected with Flag-OLFM4. After 48 hours, cell lysates were collected, and immunoprecipitation was performed using anti-Flag antibody. Western blotting was used to detect whether TRIM21 was co-precipitated. Experiment 2: HEK293T cells were transfected with HA-FGFR1, and immunoprecipitation was performed using anti-HA antibody to detect whether TRIM21 was co-precipitated. Experiment 3: A co-transfection combination of FGFR1-HA, TRIM21-His, and OLFM4-Flag was set up, and immunoprecipitation was performed using anti-HA, anti-Flag, and anti-His antibodies respectively to detect the interaction among the three. Corresponding negative controls were set up for each experiment, and Input was used as a transfection efficiency control.

[0063] 4. Experimental Results LC-MS / MS mass spectrometry results showed that 102 unique proteins were identified in the IgG group and 51 unique proteins (including OLFM4, NF2, TRIM21, EIF3B, etc.) were identified in the Flag-OLFM4 group. The number of proteins in the overlapping region between the two groups was 178. Figure 5 A). TRIM21, as an E3 ubiquitin ligase, was screened as a candidate interacting protein with OLFM4.

[0064] Immunoprecipitation validation results showed that TRIM21-specific signals were detected in the anti-Flag immunoprecipitation complex in the Flag-OLFM4 transfection group, indicating that TRIM21 can coprecipitate with OLFM4. Figure 5 B). In the HA-FGFR1 transfection group, a TRIM21-specific signal was detected in the anti-HA immunoprecipitation complex, suggesting that TRIM21 can co-precipitate with FGFR1. Figure 5 C). The above results indicate that TRIM21 interacts with both OLFM4 and FGFR1.

[0065] Example 5: OLFM4 inhibits TRIM21-mediated FGFR1 ubiquitination degradation 1. Experimental Objective This embodiment aims to investigate the regulatory role of TRIM21 in the ubiquitination and degradation of FGFR1, and the function of OLFM4 in this process.

[0066] 2. Experimental Materials Same as Example 1. MG132 (proteasome inhibitor), chloroquine (autophagy-lysosome pathway inhibitor), and HA-Ub plasmid are also required.

[0067] 3. Experimental Methods 3.1 Experiments with treatment of proteasome and lysosomal pathway inhibitors In HEK293T cells, FGFR1-HA, TRIM21-His, and OLFM4-Flag were transfected or co-transfected, respectively. Twenty-four hours after transfection, the cells were treated with either the proteasome inhibitor MG132 (10 μM) or the autophagy-lysosome pathway inhibitor chloroquine (50 μM) for six hours. Cell lysates were collected for Western blotting, using GAPDH as an internal control. Changes in band intensity before and after inhibitor treatment were compared to analyze the degradation-dependent pathway types of each protein.

[0068] 3.2 Ubiquitination Detection Experiment Ubiquitination detection experiments were performed in HEK293T cells. Experimental setup: constant overexpression of FGFR1-Myc (5 μg) and HA-Ub (5 μg), with different overexpression combinations of TRIM21-His and OLFM4-Flag. Twenty-four hours after transfection, cells were treated with MG132 (10 μM) and chloroquine (50 μM) for six hours to block protein degradation pathways, ensuring the accumulation of ubiquitinated proteins could be detected. Cell lysates were collected, and a portion was stored as input. The remaining lysate was immunoprecipitated with anti-Myc antibody to enrich FGFR1-Myc and its binding protein complex, and the eluted solution was used for Western blotting. The ubiquitination level of FGFR1 was detected using anti-HA antibody, and the expression of Flag, His, Myc, and GAPDH was also detected.

[0069] 4. Experimental Results The proteasome / lysosome inhibition assay showed that MG132 treatment significantly enhanced TRIM21 protein levels, while chloroquine treatment had a weaker effect, suggesting that TRIM21 is mainly degraded via the proteasome pathway. MG132 and chloroquine treatments had no significant effect on the band intensity of FGFR1 and OLFM4, suggesting that their degradation is weakly dependent on the proteasome and autophagy-lysosome pathways. Figure 6 A).

[0070] The ubiquitination assay results showed that, under the premise of blocking the protein degradation pathway, TRIM21 overexpression significantly promoted the polyubiquitination modification of FGFR1; while co-expression of OLFM4 and TRIM21 effectively inhibited TRIM21-mediated FGFR1 ubiquitination. Figure 6 B). The stability of total FGFR1 protein levels across different input groups suggests that OLFM4 stabilizes FGFR1 by binding to TRIM21 and inhibiting its ubiquitination and degradation. This result provides direct experimental evidence for the existence of the "OLFM4-TRIM21-FGFR1" regulatory axis.

[0071] Example 6: Validation of Clinical Tissue Samples 1. Experimental Objective This embodiment aims to verify the expression association of the OLFM4-TRIM21-FGFR1 signal axis in clinical biliary tract tumor tissues and its relationship with patient prognosis.

[0072] 2. Experimental Materials (1) Clinical sample: Biliary tract tumor tissue microarray, containing surgically removed biliary tract tumor tissue and paired adjacent normal tissue.

[0073] (2) Major antibodies: anti-OLFM4, anti-FGFR1, anti-TRIM21, anti-p-ERK1 / 2, anti-CCL2, anti-CD66b.

[0074] (3) Main reagents: Immunohistochemical staining kit, DAB chromogenic kit, hematoxylin.

[0075] (4) Main instruments: pathological slicer, slide burner, microscope, panoramic tissue scanner.

[0076] 3. Experimental Methods Immunohistochemical staining was performed on biliary tract tumor tissue microarrays. Paraffin sections were dewaxed with xylene, rehydrated with graded ethanol, and then antigen retrieval was performed using citrate buffer. Endogenous peroxidase activity was eliminated with 3% H2O2, and the sections were blocked with 10% normal goat serum. Primary antibodies against OLFM4, FGFR1, TRIM21, p-ERK1 / 2, CCL2, and CD66b were added, and the sections were incubated overnight at 4°C. The next day, after washing, HRP-labeled secondary antibody was added, and the sections were incubated at room temperature. DAB staining was performed, followed by hematoxylin counterstaining, dehydration, clearing, and mounting. The staining results were scored from 0 to 3 based on OLFM4 expression levels. Based on the scores, cases were divided into high-expression and low-expression groups, and the expression of each target marker was observed.

[0077] Based on the molecular axis activity score (GSVA score), the patient cohort was divided into a high-score group and a low-score group. The overall survival of the two groups was compared and analyzed using Kaplan-Meier survival curves combined with the Log-rank test.

[0078] 4. Experimental Results Immunohistochemical staining results showed that in the OLFM4 high-expression group, FGFR1, TRIM21, p-ERK1 / 2, CCL2, and the neutrophil marker CD66b all showed significant brownish-red positive staining, and the positive signals were mainly located in the cytoplasm of tumor cells and infiltrating immune cell regions; while in the OLFM4 low-expression group, the staining intensity of the above targets was generally weakened, and the positive signals of p-ERK1 / 2 and CCL2 were almost absent. Figure 7 The results indicate that when OLFM4 is highly expressed in tumor tissue, the expression levels of TRIM21 and FGFR1 increase synchronously, the phosphorylation level of downstream p-ERK1 / 2 and the expression of CCL2 chemokine are also significantly enhanced, accompanied by an increase in the infiltration of CD66b-positive neutrophils.

[0079] Survival analysis showed that patients with high GSVA scores had a significantly lower overall survival rate than those with low scores (p=0.018), and this survival disadvantage worsened with increasing follow-up time. By the 60-month follow-up endpoint, the survival rate of the high-score group approached zero, while the low-score group still maintained a long-term survival rate of approximately 20%. Figure 8 This result indicates that the activation state of the signal axis initiated by OLFM4 is closely related to poor prognosis in patients with biliary tract tumors.

[0080] Example 7: Working model of OLFM4-TRIM21-FGFR1 control axis Based on the results of the above embodiments, the present invention proposes a working model for the “OLFM4-TRIM21-FGFR1” control axis: When OLFM4 expression is upregulated, OLFM4 forms a complex with TRIM21, altering the catalytic activity or spatial orientation of TRIM21 and inhibiting its ubiquitination modification of FGFR1, thereby stabilizing FGFR1 protein and prolonging its signal output time. The stabilized FGFR1 further activates the downstream MAPK-AP1 signaling cascade, upregulates the expression of chemokines such as CCL2, promotes the recruitment of immature neutrophils and the formation of NETosis, reshapes the immunosuppressive microenvironment, and drives the malignant progression of tumors.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A molecular mechanism regulating the stability of FGFR1 protein, characterized in that, This is achieved by targeting the assembly of the OLFM4-TRIM21-FGFR1 ternary complex; The mechanism includes: using the competitive binding of OLFM4 protein to TRIM21 protein to inhibit TRIM21-mediated ubiquitination and degradation of FGFR1 protein, thereby stabilizing FGFR1 protein and maintaining the activation of its downstream signaling pathways.

2. The mechanism according to claim 1, characterized in that, The OLFM4 protein competitively binds to the TRIM21 protein, and its binding interface includes at least the immunoglobulin-like domain I and domain III of the FGFR1 protein.

3. A method for screening candidate substances that regulate the stability of FGFR1 protein, characterized in that, The method includes the following steps: a) Contact the analyte with a system containing OLFM4, TRIM21 and FGFR1; b) Detect the ubiquitination level or stability of FGFR1 protein in the system; and c) Compared with the control, if the test substance can promote the binding of OLFM4 to TRIM21, or inhibit the binding of TRIM21 to FGFR1, or directly reduce the ubiquitination level of FGFR1, then the test substance is identified as a candidate substance for regulating the stability of FGFR1 protein.

4. The method according to claim 3, characterized in that, The detection methods described in step b) include co-precipitation immunoassay, Western blotting, immunofluorescence, or bimolecular fluorescence complementation assay.

5. The use of OLFM4 protein or its functional fragments in the preparation of drugs or kits for stabilizing FGFR1 protein.

6. The use of TRIM21 protein or its functional fragments in the preparation of drugs or kits for promoting the degradation of FGFR1 protein.

7. The use of an inhibitor of the OLFM4 protein in the preparation of a medicament for treating diseases related to abnormal activation of the FGFR1 signaling pathway, characterized in that, The OLFM4 protein inhibitor relieves the inhibition of TRIM21-mediated FGFR1 ubiquitination degradation by blocking the binding of OLFM4 to TRIM21.

8. A pharmaceutical composition for treating diseases related to abnormal activation of the FGFR1 signaling pathway, characterized in that, The invention comprises an effective amount of the inhibitor of the OLFM4 protein as described in claim 7, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The inhibitor of the OLFM4 protein is selected from one or more of the following groups: siRNA, shRNA, antisense oligonucleotides, CRISPR gene editing systems, specific antibodies or their antigen-binding fragments, and small molecule compounds that target OLFM4.

10. Application of a biomaterial containing OLFM4, TRIM21 and FGFR1 in constructing a model for screening drugs that regulate the stability of FGFR1 protein.

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