EGFR-TKI drug resistance inhibitor and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a lung adenocarcinoma EGFR-TKI drug resistance inhibitor and application thereof. The invention firstly discloses application of a compound in preparation of a medicine for inhibiting or reversing drug resistance of an epidermal growth factor receptor tyrosine kinase inhibitor, and the compound is SIYRYacetate. The invention also provides a method for screening potential drugs for inhibiting or reversing the drug resistance of the epidermal growth factor receptor tyrosine kinase inhibitor and a kit for detecting whether a patient is resistant to the epidermal growth factor receptor tyrosine kinase inhibitor. According to the application, it is clarified for the first time that expression of EPB41L1 is regulated and controlled to be up-regulated by PD-L1 nuclear translocation through m6A epitranscription, the PD-L1 nuclear translocation and secretory protein ANGPTL4 form a compound, EGFR / HER2 dimerization and continuous activation of downstream signal channels are promoted to cause drug resistance, and a brand new theoretical basis is provided for overcoming EGFR-TKI drug resistance. Moreover, a small molecular compound SIYRY acetate is screened, and the combination of the small molecular compound SIYRY acetate and osimertinib can effectively reverse PD-L1 overexpression or ARID1A deletion mediated drug resistance, so that a breakthrough treatment strategy is provided for patients with lung adenocarcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to an EGFR-TKI drug resistance inhibitor and application thereof. BACKGROUND

[0002] Lung adenocarcinoma, as a relatively common histological type of lung cancer, has an increasing incidence year by year, which poses a serious threat to human health. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is a targeted therapy drug for epidermal growth factor receptor (EGFR) gene mutation in lung adenocarcinoma. This kind of drug can inhibit the tyrosine kinase activity of EGFR and block its downstream signaling pathway, thereby inhibiting the proliferation of tumor cells and promoting the apoptosis of tumor cells, which brings significant therapeutic effect for many lung adenocarcinoma patients, prolongs the survival period of patients and improves the quality of life. However, with the progress of treatment, the problem of EGFR-TKI drug resistance gradually emerges, which becomes the main challenge in the clinical treatment of lung adenocarcinoma.

[0003] Among the many EGFR-TKI resistance mechanisms, the formation of EGFR / HER2 heterodimers is a classic resistance mechanism. Normally, the classic activation of EGFR is the formation of EGFR homodimers under the stimulation of epidermal growth factor (EGF), the phosphorylation of the intracellular tyrosine kinase domain, and then the activation of downstream signaling pathways such as PI3K / AKT, MAPK, etc., which in turn regulate the biological behaviors of cells such as growth, proliferation, differentiation, etc. However, this activation process is finely regulated, and when the EGF action is terminated, the EGFR dimers are endocytosed, and the signal transduction is terminated, so as to maintain the stability of the intracellular environment. However, in some drug-resistant lung adenocarcinoma cells, the formation of EGFR / HER2 heterodimers breaks this balance. HER2 (human epidermal growth factor receptor 2) belongs to the human epidermal growth factor receptor family as EGFR, and has similarities in structure and function with EGFR, but also has some differences. EGFR / HER2 heterodimers have high conformational stability and low internalization rate compared with EGFR homodimers, resulting in their long-term retention on the cell membrane surface, thereby continuously activating downstream signaling pathways (such as PI3K-AKT-mTOR, RAS-RAF-MEK-ERK). This abnormal signal persistent activation state enables tumor cells to bypass the inhibition of EGFR tyrosine kinase activity by EGFR-TKI, continue to proliferate and survive, thereby leading to EGFR-TKI resistance. The mechanism of EGFR / HER2 heterodimer formation is still being further studied. From the molecular level, it may involve mutations, amplifications, changes in post-translational modification of EGFR and HER2 genes, and other factors. For example, certain specific gene mutations may change the protein structure of EGFR and HER2, increase their affinity, and promote the formation of dimers; or affect the interaction between intracellular signaling molecules and receptors, and enhance the stability of dimers. From the perspective of cell microenvironment, changes in extracellular matrix components and changes in intercellular signal transduction may also promote the formation of EGFR / HER2 heterodimers. However, these mechanisms are not yet fully clear, and many unknown factors need to be further explored. SUMMARY

[0004] In view of the key role of EGFR / HER2 heterodimer formation in EGFR-TKI resistance, clarifying its formation mechanism has become a key issue to be solved in the field of targeted therapy for lung adenocarcinoma. Only by deeply understanding this mechanism can we provide a theoretical basis for the development of new drugs. In view of the above problems to be solved in the art, the purpose of the present application is to overcome or alleviate at least one aspect of the problems and defects in the prior art.

[0005] The first aspect of the present application provides a use of a compound in the preparation of a medicament for inhibiting or reversing the drug resistance of an epidermal growth factor receptor tyrosine kinase inhibitor, and according to the L4000 compound library screening, there are also several compounds which have the same effect of blocking intermolecular binding as SIYRY acetate, thereby potentially reversing EGFR-TKI drug resistance. An exemplary drug of the compound is SIYRY acetate, and the molecular structure formula is as follows:

[0006]

[0007] Further, the medicament inhibits or reverses the drug resistance to the epidermal growth factor receptor tyrosine kinase inhibitor by inhibiting the intercombination of ANGPTL4 protein and EPB41L1 protein, and blocking the formation of EGFR / HER2 dimers.

[0008] Specifically, the inhibitor class of drugs that inhibits the intercombination of the two proteins includes: small molecule inhibitors targeting protein interaction interfaces or allosteric sites, polypeptide or peptidomimetic inhibitors, antibodies or protein inhibitors, nucleic acid aptamers, allosteric inhibitors, covalent inhibitors, compounds derived from plants or microorganisms, fragment inhibitors, molecular glue, and protein degradation targeting chimera (PROTAC).

[0009] Further, the medicament further comprises a pharmaceutically acceptable additive or excipient.

[0010] Specifically, the pharmaceutical excipients include but are not limited to cross-linking agents, flow aids, tackifiers, lubricants, humectants, fillers, excipients, solubilizers, disintegrants, etc.

[0011] The second aspect of the present application provides a method for screening potential drugs for inhibiting or reversing the drug resistance of an epidermal growth factor receptor tyrosine kinase inhibitor, specifically comprising: testing the inhibitory ability of a candidate drug on the combination of ANGPTL4 protein and EPB41L1 protein.

[0012] Further, the method further comprises the step of mixing the inhibitor with a system containing the ANGPTL4 protein and the EPB41L1 protein in vitro.

[0013] Further, the method further comprises: co-incubating the candidate drug with the ANGPTL4 protein and the EPB41L1 protein, and detecting the inhibitory ability of the candidate drug on the combination of the ANGPTL4 protein and the EPB41L1 protein.

[0014] The third aspect of the present application provides a kit for detecting whether a patient is resistant to an epidermal growth factor receptor tyrosine kinase inhibitor, the kit comprising reagents for detecting the binding level of ANGPTL4 protein and EPB41L1 protein in a test sample of the patient, wherein the binding level is positively correlated with drug resistance.

[0015] Further, the kit further comprises a standard for detecting the binding level of the ANGPTL4 protein and the EPB41L1 protein.

[0016] Specifically, the standard comprises a recombinant protein standard, a purified protein standard, a freeze-dried protein standard, a spiked control standard, and a synthetic peptide segment standard.

[0017] Further, the kit detects a human or animal subject receiving treatment with a growth factor receptor tyrosine kinase inhibitor.

[0018] The fourth aspect of the present application provides a method for reducing or interfering with the drug resistance of an epidermal growth factor receptor tyrosine kinase inhibitor in a cell line in vitro, by interfering with the binding of ANGPTL4 protein and EPB41L1 protein to inhibit the activation of downstream signaling pathways.

[0019] Specifically, the downstream signaling pathway comprises the EGFR and HER2 signaling pathways.

[0020] Advantages of the present application

[0021] The present application screens out the core target gene EPB41L1 through a series of experiments such as multi-omics analysis, and clearly determines its strong correlation with EGFR-TKI drug resistance. Then, a new drug resistance mechanism is revealed in which nuclear PD-L1 regulates the expression of EPB41L1 through m6A modification, and interacts with ANGPTL4 to activate the EGFR / HER2 signaling pathway. Finally, a series of compounds represented by small molecule compound SIYRY acetate are screened out, which can specifically block the formation of the EPB41L1-ANGPTL4 complex, inhibit EGFR / HER2 heterodimerization, and through in vitro and in vivo experiments, it is confirmed that the combination of the compound and osimertinib can significantly inhibit tumor growth and reverse the development of drug resistance. The present application provides a new target and effective inhibitor with great potential for overcoming lung adenocarcinoma EGFR-TKI drug resistance, which is expected to improve the treatment effect of patients in clinical practice and has great application value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 An experimental flowchart for exploring downstream targets of nuclear PD-L1 using a mouse lung cancer model for proteomics and multi-omics high-throughput sequencing according to the present application is shown.

[0023] Figure 2The results of in vivo mouse experiments according to this application demonstrate that targeted inhibition of PD-L1 therapy can enhance the therapeutic effect of osimertinib.

[0024] Figure 3 The Venn diagram showing the integration of multi-omics data according to this application is shown.

[0025] Figure 4 This paper presents proteomic analysis and clinical data of tumor tissue from patients with lung adenocarcinoma according to this application.

[0026] Figure 5 This application demonstrates the observation of osimertinib's IC50 after EPB41L1 overexpression or knockdown using the MTS assay. 50 Changes in values ​​(Note: "**" in the figure indicates extremely significant differences (P<0.0001)).

[0027] Figure 6 This demonstrates the use of a tumor cell clonogenic assay, according to this application, to verify the sensitivity of lung adenocarcinoma cells to osimertinib treatment.

[0028] Figure 7 The results of Western blot analysis show the effects of PD-L1 overexpression according to this application on promoting EPB41L1 expression and EGFR signaling pathway activation.

[0029] Figure 8 This paper illustrates the mechanism by which PD-L1 regulates EPB41L1 expression using immunoprecipitation sequencing of m6A-methylated RNA, according to this application.

[0030] Figure 9 The Western blot results show the verification of m6A modification involved in the regulation of EPB41L1 expression by nuclear PD-L1 according to this application.

[0031] Figure 10 The Western blot results show the effect of EPB41L1 knockdown on EGFR signaling pathway activation according to this application.

[0032] Figure 11 The Western blot results show that overexpression of EPB41L1 promotes EGFR signaling pathway activation according to this application.

[0033] Figure 12 A Venn diagram showing the results of downstream gene analysis of the MDM2 / eIF5B / PD-L1 axis regulation according to this application.

[0034] Figure 13 The results of the analysis on the correlation between ANGPTL4 and prognosis in patients with lung adenocarcinoma according to this application are shown.

[0035] Figure 14 Figure 10 shows the results of Western blotting to verify that MDM2 regulates ANGPTL4 according to the present application.

[0036] Figure 15 Figure 11 shows the results of Western blotting to verify that ANGPTL4 can inhibit the EGFR signaling pathway according to the present application.

[0037] Figure 16 Figure 12 shows the results of a colony formation experiment to verify that the expression status of ANGPTL4 affects the sensitivity of lung adenocarcinoma cells to osimertinib treatment according to the present application.

[0038] Figure 17 Figure 13 shows the results of cell IC50determination according to the present application. 50 Figure 14 shows the results of determining the effect of the expression status of ANGPTL4 on the sensitivity of lung adenocarcinoma cells to osimertinib treatment (the “**” in the figure indicates a statistically very significant difference (p<0.01)).

[0039] Figure 18 Figure 15 shows the predicted binding domain between EPB41L1 and ANGPTL4 according to molecular docking according to the present application.

[0040] Figure 19 Figure 16 shows the results of co-immunoprecipitation experiments to verify the binding between EPB41L1 and ANGPTL4 according to the present application.

[0041] Figure 20 Figure 17 shows the results of co-immunoprecipitation experiments to verify the binding between EPB41L1 with a hemagglutinin tag and ANGPTL4 with a flag tag according to the present application.

[0042] Figure 21 Figure 18 shows the results of proteomic sequencing analysis of lung adenocarcinoma patient tissues according to the present application.

[0043] Figure 22 Figure 19 shows the results of Western blotting to show the expression changes of downstream pathway proteins after knocking down EPB41L1 according to the present application.

[0044] Figure 23 Figure 20 shows the results of Western blotting to show the expression changes of downstream pathway proteins after knocking down ANGPTL4 according to the present application.

[0045] Figure 24 Figure 21 shows the results of SDS-PAGE electrophoresis after purifying ANGPTL4, EPB41L1, HER2 and EGFR proteins with a GST tag or a His tag according to the present application.

[0046] Figure 25GST-pulldown results showing the interaction of GST-ANGPTL4 with His-EPB41L1 according to the present application.

[0047] Figure 26 GST-pulldown results showing the interaction of HER2 or EGFR protein with EPB41L1 and ANGPTL4, respectively, according to the present application.

[0048] Figure 27 EPB41L1 and ANGPTL4 with HER2 protein, respectively, according to the present application.

[0049] Figure 28 Schematic diagram showing the formation mechanism of the membrane protein complex EGFR / HER2 / EPB41L1 / ANGPTL4 according to the present application.

[0050] Figure 29 Simulation diagram showing the competition of the compound for binding EPB41L1 with ANGPTL4 according to the present application.

[0051] Figure 30A Comparison results of fluorescence resonance energy transfer showing the blocking of the binding of ANGPTL4 with EPB41L1 by SIYRY acetate and HAEGTFT acetate according to the present application.

[0052] Figure 30B Comparison results of fluorescence resonance energy transfer showing the blocking of the binding of ANGPTL4 with EPB41L1 by substance P and non-specific peptide (Nonapeptide) according to the present application.

[0053] Figure 31 Immunoprecipitation results showing the blocking of the pairwise interaction among EGFR / HER2 / EPB41L1 by SIRYR acetate according to the present application.

[0054] Figure 32 Western blot results showing the blocking of the pairwise interaction among EGFR / HER2 / EPB41L1 by SIRYR acetate according to the present application.

[0055] Figure 33 Western blot results showing that ARID1A knockdown promotes the expression of PD-L1 and EGFR signaling pathway activation proteins according to the present application.

[0056] Figure 34Figure showing the simulation effect of the molecular docking of SIRYR acetate on the EGFR / HER2 / EPB41L1 / ANGPTL4 protein tetramer action site according to the present application.

[0057] Figure 35 Figure showing the Western blot results of the addition of exogenous recombinant ANGPTL4 protein according to the present application to restore the EGFR signaling pathway inactivated due to ANGPTL4 knockdown.

[0058] Figure 36 Figure showing the Western blot results of the addition of SIYRY acetate treatment in a PD-L1 overexpressing cell line according to the present application to inhibit the activation of the EGFR signaling pathway caused by changes in gene expression and reduce HER2 expression.

[0059] Figure 37 Figure showing the Western blot results of the addition of SIYRY acetate treatment in an ARID1A knockdown cell line according to the present application to inhibit the activation of the EGFR signaling pathway caused by changes in gene expression and reduce HER2 expression.

[0060] Figure 38 Figure showing the in vivo mouse experiment according to the present application that confirms that SIYRY acetate combined with osimertinib treatment can reverse the drug resistance phenomenon that occurs due to ARID1A knockdown.

[0061] Figure 39 Figure showing the flowchart of the overall research plan according to the present application. DETAILED DESCRIPTION

[0062] TERMS DEFINITION

[0063] As used herein, "Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance" refers to the phenomenon of tumor cells developing resistance to treatment with tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR). For example, non-small cell lung cancer (NSCLC) patients carrying EGFR (epidermal growth factor receptor) mutations, after initial sensitivity to EGFR tyrosine kinase inhibitors (such as gefitinib, erlotinib, osimertinib, etc.) treatment, tumor cells escape drug action through various mechanisms, leading to the phenomenon of disease progression.

[0064] As used herein, "Input" in Co-Immunoprecipitation (Co-IP) experiments generally refers to the total protein sample added at the beginning of the experiment (lysate before immunoprecipitation), that is, the total protein mixture extracted from cells or tissues before Co-Immunoprecipitation. It represents the initial total amount of protein available for Co-Immunoprecipitation in the experiment. It is used to prove the original presence of target proteins in the sample to exclude experimental false negatives, as a quantitative reference to verify the efficiency of immunoprecipitation, and to detect antibody specificity by comparison with samples after IP.

[0065] As used herein, "MDM2 knockdown cell line" is a cell model in which the expression of the MDM2 (Mouse Double Minute 2) gene is reduced or silenced by genetic engineering techniques such as RNA interference or CRISPR-Cas9, mainly used to study the role of MDM2 in tumorigenesis, apoptosis regulation and p53 signaling pathway, where MDM2 is an E3 ubiquitin ligase that negatively regulates the activity of p53 (tumor suppressor protein) by ubiquitination.

[0066] Research background of the present application

[0067] Lung cancer is the most common cause of cancer-related mortality, with non-small cell lung cancer (NSCLC) accounting for nearly 85% of lung cancer cases, and lung adenocarcinoma (LUAD) being one of the main types of NSCLC. ARID1A gene, as a key subunit of SWI / SNF chromatin remodeling complex, is involved in DNA repair and stabilization. Mutations in this gene have been found in various cancers and are associated with tumor invasiveness, immune escape, and synthetic lethality. Loss of ARID1A expression in LUAD is associated with tumor proliferation and apoptosis inhibition, but its role in LUAD invasion and metastasis is not yet clear. Sun D et al. found that ARID1A knockdown upregulated p-Akt expression by activating the Akt signaling pathway, which may promote LUAD metastasis. GSEA analysis also supports the activation of the Akt signaling pathway in patients with low ARID1A expression. Therefore, loss of ARID1A expression can be used as a marker for poor prognosis in LUAD patients. ARID1A may serve as a new target for LUAD treatment, and its expression level can be used to predict the prognosis of LUAD patients and guide clinical treatment decisions. Sun D, Zhu Y, Zhao H, Bian T, Li T, Liu K, Feng L, Li H, Hou H. Loss of ARID1A expression promotes lung adenocarcinoma metastasis and predicts a poor prognosis. Cell Oncol (Dordr). 2021; 44(5): 1019-1034. Another study found that ARID1A acts as a tumor suppressor in EGFR-mutated LUAD, and its loss activates multiple oncogenic pathways, leading to accelerated cell cycle, increased cell division, and enhanced metastatic ability. Low ARID1A expression is associated with poor response to first-generation EGFR-TKI therapy in patients with EGFR-mutated LUAD, and may serve as a biomarker for predicting resistance to EGFR-TKI therapy.The study also proposed potential therapeutic strategies targeting the PI3K / Akt / mTOR pathway and multi-target TKI, which may be effective for ARID1A-deficient NSCLC patients ([2] Sun D, Feng F, Teng F, Xie T, Wang J, Xing P, Qian H, Li J. Multiomics analysis revealed the mechanisms related to the enhancement of proliferation, metastasis and EGFR-TKI resistance in EGFR-mutant LUAD with ARID1A deficiency. Cell Commun Signal. 2023, 21(1):48.). Other studies have also found that mutations or loss of expression of ARID1A can lead to resistance to EGFR-TKI through various tumor development processes, including epithelial-mesenchymal transition (EMT), tumor angiogenesis, and inhibition of apoptosis. Therefore, it is speculated that changes or loss of expression of ARID1A may be a modulator of the PI3K / Akt, JAK / STAT, and NF-κB signaling pathways, which are closely related to resistance to EGFR-TKI in NSCLC patients. Based on the key role of ARID1A in EGFR-TKI resistance in non-small cell lung cancer (NSCLC), its mutations or loss of expression lead to resistance through multiple mechanisms, therapeutic strategies targeting ARID1A or related signaling pathways may provide new ideas for overcoming EGFR-TKI resistance ([3] Sun D, Teng F, Xing P, Li J. ARID1A serves as a receivable biomarker for the resistance to EGFR-TKI in non-small cell lung cancer. Mol Med. 2021, 27(1):138.). Based on the above research results, further research on the specific mechanisms of ARID1A deficiency-mediated EGFR-TKI resistance has been conducted, confirming that the loss of ARID1A is significantly associated with osimertinib resistance. Patients with ARID1A deficiency had significantly shorter median progression-free survival (PFS) after osimertinib treatment than patients with high ARID1A expression (6.20 months vs 11.20 months, P=0.0183). The reason is that ARID1A deficiency promotes the nuclear translocation of PD-L1 and the activation of the Ras signaling pathway by affecting the EZH2 / PTEN / E2F1 axis and the MDM2 / eIF5B / PD-L1 axis, inhibiting autophagy and apoptosis of tumor cells, and promoting tumor progression and osimertinib resistance.The molecular mechanism is the increase of PD-L1 expression due to ARID1A deletion, and promotes its nuclear translocation. After nuclear translocation, PD-L1 (nPD-L1) interacts with CD44 to form a complex, activates the RASGEF1A promoter, starts the Ras signaling pathway, and leads to osimertinib resistance. Sun D et al. also proposed a treatment strategy, by targeting the transcription, translation and nuclear translocation of PD-L1, the osimertinib resistance caused by ARID1A deletion can be overcome. Using lipid nanoparticles (LNP) to deliver small interfering RNA (siRNA) against PD-L1 can effectively inhibit the expression of PD-L1 and the activation of the Ras signaling pathway, and enhance the efficacy of osimertinib. ([4] Sun D, Hou H, Feng F, Wu W, Tan J, Xie T, Liu J, Wang J, Qian H, Li J, Xing P. A cohort-based multi-omics identifies nuclear translocation of eIF5B / PD-L1 / CD44 complex as the target to overcome Osimertinib resistance of ARID1A-deficient lung adenocarcinoma. Exp Hematol Oncol. 2025 Jan 7;14(1):3).

[0068] Based on the above findings that ARID1A deletion causes EGFR-TKI (such as osimertinib) resistance, the research strategy of the present application is designed as follows:

[0069] 1. Proteomics detection of lung cancer tissue combined with sequencing for PD-L1 to find downstream target genes of nPD-L1.

[0070] 2. Find the way in which the expression changes of the downstream target genes of PD-L1 affect the sensitivity of lung adenocarcinoma cells to EGFR-TKI treatment.

[0071] 3. Explore the regulation principle and action mode of nPD-L1 on target genes.

[0072] 4. Find EGFR-TKI resistance related genes interacting with the target gene, and verify the interaction between the two.

[0073] 5. Explore the interaction mechanism between the EGFR-TKI resistance related genes and the EGFR / HER2 heterodimeric protein.

[0074] 6. According to the corresponding interaction mechanism, screen for compounds that can verify the interaction mechanism.

[0075] 7. In vivo and in vitro verification of whether the compound can effectively inhibit the interaction between EGFR-TKI resistance related genes and HER2 protein, thereby reducing or eliminating the EGFR-TKI resistance phenomenon caused by the formation of EGFR / HER2 heterodimers.

[0076] In order for those skilled in the art to have a clearer understanding of the technical solutions of the present application, the following examples and test examples will be further described in detail. It should be noted that the examples described are only a part of the present application, not all. On the basis of the present application, those skilled in the art can imitate without creative labor, and other examples obtained are within the protection scope of the present application. A series of experimental techniques and experimental methods involved and adopted in the present application, if not specially marked or additionally explained, are all default to follow the conventional technical path and operation specification recognized in the field. Specifically, each experimental operation including but not limited to molecular biology experiment, microbial culture and identification, biochemical analysis, etc. is executed according to the standard process in authoritative textbooks and industry guidelines. For example, for the experimental steps without clear marked specific operation conditions, usually according to the conventional laboratory conditions, which are recorded in detail in classic works such as "Molecular Cloning Experiment Guide (3rd Edition)" (Science Press) and "Microbiology Experiment (4th Edition)" (Higher Education Press), and also can follow the official recommended conditions provided by the manufacturers of reagents, instruments and consumables used in the experiment. In the selection of experimental materials and reagents, unless otherwise specified in the present embodiment, all biological materials (such as strains, cell lines, etc.), chemical reagents (including but not limited to buffer, enzyme preparation, antibody, etc.) and experimental consumables (such as culture dishes, centrifuge tubes, pipette gun heads, etc.) used are purchased through legal and compliant commercial channels, to ensure that their quality meets the experimental requirements and industry standards.

[0077] All reagents and instruments used in the examples are shown in Tables 1-2

[0078] Table 1 Main instruments and equipment

[0079] Instrument name Company Vacuum concentrator Thermo-Fisher Scientific Quadrupole mass spectrometer Thermo-Fisher Scientific IncuCyte live cell imager Sartorius Transmission electron microscope Hitachi Laser confocal microscope Nikon FTC-3000p real-time PCR system Funglyn Biotech LNP preparation chip Shanghai Pengzan Biotech Dual-channel syringe pump Nanjing Ximai Nanometer Technology Ultramicrotome Leica

[0080] Table 2 Main reagents

[0081] Antibody Company Antibody Company ARID1A Abcam PD-L1 PROTEINTECH EPB41L1 PROTEINTECH CD44 Cell Signaling Technology ANGPTL4 PROTEINTECH MDM2 Cell Signaling Technology mTOR Cell Signaling Technology HA PROTEINTECH phosphorylated-MAPK Cell Signaling Technology phosphorylated-HER2 Cell Signaling Technology STAT3 Cell Signaling Technology pan-AKT Cell Signaling Technology phosphorylated-STAT3 Cell Signaling Technology phosphorylated-AKT Cell Signaling Technology β-actin Sigma-Aldrich MAPK Cell Signaling Technology PCNA PROTEINTECH TP53 Cell Signaling Technology phosphorylated-mTOR Cell Signaling Technology PARP Cell Signaling Technology EGFR Cell Signaling Technology FTO PROTEINTECH phosphorylated-EGFR Cell Signaling Technology Ras Cell Signaling Technology HER2 PROTEINTECH FLAG PROTEINTECH eIF5B SANTACRUZ

[0082] Example 1 Exploration of downstream action target points after nuclear translocation of PD-L1

[0083] 1. It is proved by mouse in vivo experiment that the treatment of targeting inhibition of PD-L1 nuclear translocation can enhance the therapeutic effect of osimertinib

[0084] Using lipid nanoparticles (LNP) to encapsulate si-RNA against PD-L1, the si-RNA was delivered into tumor cells to inhibit the expression of PD-L1 and block nuclear translocation (see Experimental Procedures in Figure 1 ). This LNP can effectively deliver si-RNA into tumor cells for the treatment of lung cancer mouse models, (see Acohort-based multi-omics identifies nuclear translocation of eIF5B / PD-L1 / CD44 complex as the target to overcome Osimertinib resistance of ARID1A-deficient lung adenocarcinoma. Exp Hematol Oncol. 2025 Jan 7; 14(1): 3 (see Additional Materials and Methods section of the supplementary materials of the literature [4], see Additional 1 associated data, specifically page 15, line 4, 26. In vivo xenograft model, download link: https: / / pmc.ncbi.nlm.nih.gov / articles / PMC11705878 / #_ad93) for specific materials and methods such as si-RNA sequence, LNP encapsulated si-RNA and administration method). The results showed that targeting PD-L1 can enhance the efficacy of EGFR-TKI, Figure 2 The experimental results showed that LNP / si-RNA inhibiting PD-L1 synthesis can enhance the efficacy of Osimertinib.

[0085] Figure 2 The relative change in tumor volume within 25 days for different treatment groups (dimethyl sulfoxide (DMSO), Osimertinib, Osimertinib + LNP si-Control, Osimertinib + LNP si-PD-L1) is shown in Figure Figure 2 The results showed that this treatment method can significantly enhance the efficacy of Osimertinib, and slow down the growth of tumors by blocking the nuclear translocation of PD-L1.

[0086] 2. Detection of downstream target genes of PD-L1 by proteomics and high-throughput sequencing

[0087] The tumor tissues collected from the above two groups of mice (osimertinib+LNP si-Control, osimertinib+LNP si-PD-L1) were subjected to proteomics detection (Shanghai iProteome Biotechnology Co., Ltd.), combined with RNA-seq (RNA sequencing, gene expression changes in ARID1A deletion cells were analyzed by RNA-seq to find downstream target points related to PD-L1), RIP-seq (RNA immunoprecipitation sequencing, the RNA binding of PD-L1 in cells was analyzed by RIP-seq to determine whether PD-L1 directly interacts with specific mRNA), and ATAC-seq (transposase accessible chromatin sequencing, the effect of PD-L1 on chromatin accessibility was analyzed by ATAC-seq to find the gene promoter region that PD-L1 may regulate) for high-throughput sequencing (Wuhan Kangmei Technology Co., Ltd.), and the four technologies of RNA-seq, RIP-seq, proteomics and ATAC-seq were used to screen the key downstream genes of EPB41L1 regulated by nuclear PD-L1.

[0088] Figure 3 The Venn diagram result of multi-omics data integration analysis, wherein the numbers represent the number of different genes / proteins / sites regulated or affected by PD-L1 screened by each sequencing technology, and the intersection relationship is shown by Venn diagram. Specifically, the RNA-seq sequencing result shows that 94 genes are related to the expression of PD-L1. The RIP-seq sequencing result shows that 56 RNA molecules are directly combined with PD-L1. The proteomics research result shows that among 3715 proteins, 21 are related to PD-L1. The ATAC-seq sequencing result shows that 329 chromatin open regions are related to the regulation of PD-L1. Through intersection analysis, it can be known that the RNA-seq+RIP-seq sequencing result shows that 10 genes may be regulated by PD-L1 after transcription. The results of integrating RNA-seq+proteomics show that about 72 genes are involved in the common changes of transcription and protein level. Further, the RIP-seq+proteomics research result shows that only 2 molecules are involved in direct binding and protein interaction. The common intersection of each study is only 1 molecule, which is most likely the core downstream target point of PD-L1, which is EPB41L1. Therefore, it is preliminarily determined that the downstream target gene of PD-L1 is EPB41L1.

[0089] Example 2 discusses the correlation between EPB41L1 and osimertinib resistance

[0090] 1. Investigate the influence of EPB41L1 expression state on the treatment effect of osimertinib

[0091] Osimertinib is the third generation of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) that is effective for multiple EGFR-sensitive mutations and T790M drug-resistant mutations. However, some patients will have poor prognosis during the treatment with osimertinib, and the underlying mechanism has not been fully elucidated. Therefore, 101 patients with lung adenocarcinoma confirmed by pathology were selected, and their tumor tissue samples were collected. All patients signed the informed consent form before the study, and the research protocol was approved by the hospital ethics committee (Beijing University First Hospital Ethics No.: No. 2025R0066-0002; China Academy of Medical Sciences Cancer Hospital Ethics No.: No. NCC-007421). Proteomics detection was performed on the tumor tissues of 101 patients with lung adenocarcinoma, and clinical information was matched, such as collecting detailed clinical information of patients, including age, gender, smoking history, pathological stage, EGFR gene mutation status, previous treatment history, osimertinib treatment plan, treatment response (such as partial remission, stable, progression, etc.), progression-free survival (PFS), overall survival (OS), etc. Proteomics technology is used to systematically analyze protein expression in cells or tissues in order to find biomarkers related to disease development and treatment prognosis. Figure 4 Based on the survival analysis results of proteomics, the effect of EPB41L1 expression level on the progression-free survival (PFS) of lung adenocarcinoma patients treated with osimertinib was evaluated. The PFS hazard ratio (HR) of the high expression group of EPB41L1 was 2.30 (95% CI: 1.37-3.84), indicating that the risk of disease progression in patients with high expression of EPB41L1 was 2.3 times that of the low expression group. P value = 1.2e-3 (i.e. 0.0012), with high statistical significance, indicating that high expression of EPB41L1 is significantly associated with poor prognosis. In terms of the number of patients at risk (Number at risk), the number of surviving patients at each follow-up time point (24, 36, 48 months) gradually decreased, which is consistent with the survival trend of the clinical cohort. This shows that: Figure 4 The median PFS (months) of the high / low EPB41L1 expression groups is 8.54 and 16.57, respectively. The narrow confidence interval (1.37-3.84) and low P value (0.0012) indicate that the detection results are stable and not affected by random factors. According to the conclusion obtained from the above proteomics analysis, high expression of EPB41L1 is associated with poor prognosis of osimertinib, and it is an independent predictor of osimertinib resistance (HR = 2.30, P = 0.0012).

[0092] 2. Verify whether the expression status of EPB41L1 can affect the sensitivity of lung adenocarcinoma cells to osimertinib treatment

[0093] Based on the above sequencing and proteomics results, verification is needed from the level of cellular proteins. Therefore, the cell line IC50 Phenotype validation (experimental methods see the supplementary materials and methods section of [4], page 14, line 25, 25. MTS assay).

[0094] Figure 5 IC of EPB41L1 50 The determination results show the sensitivity of each cell to osimertinib after EPB41L1 knockdown (EPB41L1_kd) compared with the negative control group (Negative control). The IC of the EPB41L1_kd group is significantly reduced (column height 1.67), indicating that the cell is more sensitive to the drug after knocking down EPB41L1. 50 The IC of the control group is higher (column height 7.41), and there is a very significant difference between the two (indicated by “****” in the figure, indicating a very significant difference (P<0.0001)), indicating that high expression of EPB41L1 leads to drug resistance. 50 The statistical significance of the IC of EPB41L1 Figure 5 demonstrates that the expression level of EPB41L1 is directly related to the sensitivity of osimertinib.

[0095] Clonogenic assay Figure 6 compared the clonogenic ability of the EPB41L1 high expression group and the control group after treatment with different concentrations of osimertinib (1 μM, 2 μM, 5 μM). Among them, the number of clones of EPB41L1 high expression cells in the DMSO group (without drug) was similar to that of the control group, indicating that high expression itself does not affect the basic proliferation. Through the results of the osimertinib treatment group, it was found that when 1 μM of osimertinib was used, EPB41L1 high expression cells still formed more clones (significant drug resistance). When the concentration of osimertinib increased to 5 μM, the cell clones of the control group almost disappeared, while the EPB41L1 high expression cells still had a small amount of surviving cell clones, indicating that drug resistance was still ongoing.

[0096] In summary, the IC 50 determination experiment and the results of the tumor cell clonogenicity experiment show that the IC 50 is significantly increased ( Figure 5 ) and the clonogenicity is enhanced ( Figure 6 ), both of which together confirm that high expression of EPB41L1 significantly reduces the sensitivity of cells to osimertinib, and ultimately it can be concluded that EPB41L1 is a key regulatory factor of osimertinib resistance, and high expression of EPB41L1 leads to drug resistance of tumor cells to osimertinib by stabilizing the EGFR signaling pathway. The molecular mechanism may be that EPB41L1 drives drug resistance by promoting EGFR / HER2 dimerization or nuclear PD-L1 signaling pathway.

[0097] Example 3: Explore the regulation mode of nuclear PD-L1 on EPB41L1 expression

[0098] The results of the above Examples 1 and 2 demonstrate that the expression status of EPB41L1 can affect the sensitivity of lung adenocarcinoma cells to osimertinib treatment, and high expression of EPB41L1 can lead to drug resistance. This example further clarifies the regulation mode of nuclear PD-L1 on EPB41L1 by methylated RNA immunoprecipitation sequencing (meRIP-seq) detection (Wuhan Kangmei Technology Co., Ltd.).

[0099] The results of the Western blot experiment Figure 7 ) show the expression levels of various proteins, where Ctr is the control group and PD-L1 is the treatment group. Changes in the expression levels of various proteins can be observed after treatment, including PD-L1, EPB41L1, EGFR and its phosphorylated form, mTOR pathway proteins and their phosphorylated forms, CD44, FTO (m6A demethylase), and β-actin (β-Actin), which serves as an internal reference protein and is uniformly expressed in each sample, indicating consistent loading amounts and reliable results. In the PD-L1 treatment group, PD-L1 expression is significantly upregulated, indicating that the experimental treatment successfully induced PD-L1 expression. The expression level of EPB41L1 shows a positive correlation with PD-L1, with EPB41L1 expression increasing when PD-L1 is highly expressed, confirming the speculation that PD-L1 positively regulates EPB41L1. The Western blot results of EGFR and p-EGFR show that total EGFR expression is relatively stable, while the activity of p-EGFR (phosphorylated EGFR) increases significantly, indicating that PD-L1 stimulates the activation of the EGFR signaling pathway. The blot results of mTOR and p-mTOR are similar to those of EGFR / p-EGFR, with changes in p-mTOR activity related to PD-L1 expression, suggesting that the mTOR pathway is involved in the downstream effects of PD-L1. Changes in the expression level of the cell surface glycoprotein CD44 indicate changes in cell stemness or migration ability. The expression of m6A demethylase (FTO) increases slightly, indicating that PD-L1 is a key factor affecting m6A modification.

[0100] Subsequently, m6A methylated RNA immunoprecipitation sequencing (m6A meRIP-seq) was performed to investigate whether the upregulation of EPB41L1 expression by nuclear translocation of PD-L1 is related to m6A modification. Figure 8The results are as follows. The top figure quantifies the changes in the proportion of m6A modification. The outer circle shows the chromosome number, with each chromosome represented by a different color. The inner circle shows that m6A modification is more concentrated in certain chromosomal regions, which may be related to gene expression regulation. The bottom figure shows the m6A modification status of the EPB41L1 gene. The figure shows the m6A modification levels in both Input and immunoprecipitation (IP) samples. The horizontal axis of the bar chart represents genomic location, and the vertical axis represents the signal intensity of m6A modification. Significant m6A modification signals are visible in the EPB41L1 gene region, especially in the control group samples, where these signals are more pronounced. Figure 8 As can be seen, PD-L1 overexpression treatment weakened the m6A modification level of the EPB41L1 gene. This indicates that PD-L1 affects the expression or function of the EPB41L1 gene by regulating m6A modification. High PD-L1 expression enhances EPB41L1 protein expression by weakening m6A modification of EPB41L1 mRNA (top figure) (bottom figure), indicating that nuclear PD-L1 upregulates EPB41L1 gene expression by adjusting m6A epigenetic transcriptional regulation.

[0101] Example 4 verifies the role of m6A modification in regulating the expression of nuclear PD-L1 on EPB41L1.

[0102] Analysis in Example 3 showed that PD-L1 can affect the expression or function of the EPB41L1 gene by regulating m6A modification. This example further utilizes FB23-2 (an FTO inhibitor that can directly bind to FTO and selectively inhibit its m6A modification). 6 Cells were treated with m6A demethylation activity and Western blot was used to verify that m6A modification was involved in the regulation of nuclear PD-L1 expression of EPB41L1 (for specific experimental methods, please refer to the supplementary materials and methods section of the above reference [4], page 10, line 30, 17.WB).

[0103] in Figure 9 The results showed that the control group (--) was untreated (basal expression level), the PD-L1 treatment group (+) was PD-L1 high expression, and PD-L1+FB23-2 (10μM) was PD-L1 high expression + FB23-2 (10μM m6A methylation inhibitor) treatment for 24 hours. Figure 9 As can be seen, PD-L1 ( +) significantly increased EPB41L1 expression (supporting PD-L1 positive regulation of EPB41L1), PD-L1+FB23-2 decreased EPB41L1 expression (close to the control group level). It is shown that FB23-2 inhibition of m6A modification reverses the promotion and stimulation of PD-L1 on EPB41L1, which directly proves that m6A modification is involved in the regulation of PD-L1 on EPB41L1. It is also found that the trend of the change of angiopoietin-like protein ANGPTL4 is similar to that of EPB41L1, indicating that the gene is regulated by a common mechanism. EGFR / p-EGFR, HER2 / p-HER2 are PD-L1 activated receptor tyrosine kinase (RTK) signals, and FB23-2 partially inhibits the phosphorylation effect. AKT / p-AKT, mTOR / p-mTOR and a series of AKT-mTOR pathway signals activated by PD-L1, FB23-2 can weaken their phosphorylation. The results of Western blot of MAPK / p-MAPK, Ras show the activation of MAPK and Ras signaling pathway by PD-L1, and the activity decreases after FB23-2 intervention. At the same time, PD-L1 up-regulates the stem cell marker CD44, and FB23-2 inhibits its expression, suggesting that m6A modification affects tumor stemness. The above results confirm that m6A modification is indeed involved in the regulation of nuclear PD-L1 on the expression of EPB41L1.

[0104] Example 5 verifies whether EPB41L1 can activate the EGFR signaling pathway

[0105] According to the results of the above Example 4, the Western blot results were further explored to investigate whether EPB41L1 can affect the activation of the EGFR signaling pathway and further affect drug resistance, and the results are as follows Figure 10 and Figure 11 . Figure 10 is the protein expression of sh-EPB41L1 and sh-Ctrl cells. In the sample of EPB41L1 knockdown, the expression of key proteins in the EGFR signaling pathway changed compared with the control group. Among them, the expression levels of p-EGFR (phosphorylated EGFR) and p-mTOR (phosphorylated mTOR) were significantly reduced after EPB41L1 knockdown, indicating that EPB41L1 positively regulates the activation of these proteins. The expression levels of other proteins such as EGFR, mTOR, PCNA, and RAS also showed similar changes. Figure 11 is the protein expression of sh-EPB41L1 and sh-Ctrl cells. In the sample of EPB41L1 knockdown, the expression of key proteins in the EGFR signaling pathway changed compared with the control group. Among them, the expression levels of p-EGFR (phosphorylated EGFR) and p-mTOR (phosphorylated mTOR) were significantly reduced after EPB41L1 knockdown, indicating that EPB41L1 positively regulates the activation of these proteins. The expression levels of other proteins such as EGFR, mTOR, PCNA, and RAS also showed similar changes. Figure 10The results were contrary. The expression levels of the above proteins such as p-EGFR and p-mTOR were significantly increased after overexpression of EPB41L1, further supporting that EPB41L1 positively regulates the activation of these proteins. The experimental results show that EPB41L1 can affect the activation of the EGFR signaling pathway, especially the key proteins in the pathway such as phosphorylated EGFR (p-EGFR) and phosphorylated mTOR (p-mTOR) are significantly positively regulated by EPB41L1.

[0106] Example 6 Research on the mechanism of ANGPTL4 and EPB41L1 jointly mediating EGFR-TKI drug resistance

[0107] It is found from the research results of Example 4 that the change trend of angiopoietin-like protein ANGPTL4 is similar to that of EPB41L1, and it is suspected that the gene is regulated by a common mechanism. Therefore, this embodiment will further explore the relationship between the two molecules of EPB41L1 and ANGPTL4.

[0108] Firstly, through RNA-seq of MDM2 knockdown cell line MDM2_kd, RIP-seq of eIF5B and PD-L1 protein (Wuhan Konbio Technology Co., Ltd.), the regulation network of MDM2-eIF5B-PD-L1 molecular axis on downstream genes was studied, and the direct target genes of the molecular axis were screened. A group of downstream genes directly regulated by the MDM2 / eIF5B / PD-L1 axis was obtained. Venn diagram Figure 12)Specifically, it shows the overlap of gene sets obtained by MDM2 knockdown (MDM2_kd), eIF5B RNA immunoprecipitation (eIF5B RIP), and PD-L1 RNA immunoprecipitation (PD-L1 RIP). Among them, MDM2_kd (green circle) represents the differentially expressed genes obtained by RNA-seq experiment in MDM2 knockdown cell lines, a total of 514 genes. eIF5B (blue circle) represents the genes directly interacting with eIF5B obtained by eIF5B RNA immunoprecipitation (RIP-seq) experiment, a total of 1402 genes. PD-L1 (red circle) represents the genes directly interacting with PD-L1 obtained by PD-L1 RNA immunoprecipitation (RIP-seq) experiment, a total of 2562 genes. The overlap between MDM2_kd and eIF5B is 42 genes, which are identified in both MDM2 knockdown and eIF5B RIP-seq. The overlap between MDM2_kd and PD-L1 is 210 genes, which are identified in both MDM2 knockdown and PD-L1 RIP-seq. In addition, the overlap between eIF5B and PD-L1 is 2029 genes, which are identified in both eIF5B RIP-seq and PD-L1 RIP-seq. The common overlap of the three is 29 genes, which are identified in MDM2 knockdown, eIF5B RIP-seq and PD-L1 RIP-seq. These genes are considered to be directly regulated downstream genes of the MDM2 / eIF5B / PD-L1 axis. The identification of these genes reveals the potential synergistic effect of MDM2, eIF5B and PD-L1 in regulating gene expression. In particular, those 29 genes that appear in all three high-throughput sequencing are key targets of this regulatory axis, which play an important role in cell biology processes.

[0109] After obtaining the above important genes, it was found that ANGPTL4 was most related to the prognosis of lung adenocarcinoma patients according to the GEPIA database (http: / / gepia.cancer-pku.cn / ). As shown in Figure 13 , patients with high expression of ANGPTL4 had shorter overall survival (OS, HR = 1.7, p = 0.0079) and disease-free survival (DFS, HR = 1.5, p = 0.0012), suggesting that ANGPTL4 can be used as an independent poor prognostic marker for lung adenocarcinoma.

[0110] will be described in detail Figure 13which collectively comprises two parts: the left side is an Overall Survival curve and the right side is a Disease Free Survival curve. In the Overall Survival curve, the horizontal axis represents time (months) and the vertical axis represents survival percentage. The survival curves of two groups of patients are shown in the left graph, one group is patients with low expression level of ANGPTL4 (Low ANGPTL4 TPM) and the other group is patients with high expression level of ANGPTL4 (High ANGPTL4 TPM). It can be seen from the curve that the overall survival rate of patients with low expression level of ANGPTL4 is higher, while the overall survival rate of patients with high expression level of ANGPTL4 is lower. From the statistical data in the left graph, the Log rank p value (p = 0.00068), the hazard ratio (HR) = 0.00079, and the sample size of the two groups (n(high) = 239, n(low) = 239) can be seen, which indicates that the expression level of ANGPTL4 is significantly related to the overall survival.

[0111] The right graph is a Disease Free Survival curve. Similarly, the horizontal axis represents time (months) and the vertical axis represents survival percentage. The survival curves of two groups of patients are shown, patients with low expression level of ANGPTL4 have higher disease-free survival rate, while patients with high expression level of ANGPTL4 have lower disease-free survival rate.

[0112] The statistical data show that the Log rank p value (p = 0.0044), the hazard ratio (HR) = 0.0047, and the sample size of the two groups (n(high) = 239, n(low) = 239), which also indicates that the expression level of ANGPTL4 is significantly related to the disease-free survival. Overall, lung adenocarcinoma patients with high expression level of ANGPTL4 show poor overall survival and disease-free survival, which indicates that ANGPTL4 is an important indicator of poor prognosis for lung adenocarcinoma patients.

[0113] Subsequently, it was verified by western blot experiment whether MDM2 (Mouse Double Minute 2 homolog) has a regulatory effect on ANGPTL4. At the same time, it was investigated whether the use of MDM2 inhibitor Nutlin-3 can reverse the related function of MDM2. The specific results are shown in Figure 14 Figure 14 The results include three groups: the control group (-) is the untreated basic expression level, the MDM2 overexpression group (+) is the high expression condition of MDM2, and the MDM2 + Nutlin-3 (40 μM) is the high expression condition of MDM2 with the addition of MDM2 inhibitor Nutlin-3.​

[0114] From the western blot bands, with β-Actin as the internal reference protein, its expression remained stable in each group, indicating that the protein loading amount was relatively consistent, in the sample overexpressing MDM2 only, the expression of MDM2 was significantly increased, while in the sample using Nutlin-3 at the same time, the expression of MDM2 was inhibited to a certain extent. In the sample overexpressing MDM2 only, the expression of ANGPTL4 was increased, while in the sample using Nutlin-3 at the same time, the expression of ANGPTL4 was significantly reduced, which indicated that MDM2 could positively regulate the expression of ANGPTL4, and Nutlin-3 could reverse this regulation. It can be concluded that MDM2 has a positive regulation on ANGPTL4.

[0115] In order to verify whether ANGPTL4 can affect the activation of EGFR signaling pathway like EPB41L1. The present application again uses western-blot experiment to verify the influence of ANGPTL4 on EGFR signaling pathway, whether the key proteins in the pathway such as phosphorylated EGFR and phosphorylated mTOR are regulated by ANGPTL4. The protein expression of control group (sh-Ctrl) and ANGPTL4 knockdown group (sh-ANGPTL4) is shown in Figure 15 As the expression of internal reference protein β-Actin remained stable in each group, it was proved that the protein loading amount was relatively consistent, in the sample knocking down ANGPTL4, the expression of ANGPTL4 was significantly reduced. Correspondingly, the expression of EGFR signaling pathway related proteins, EGFR and p-EGFR (phosphorylated EGFR) in the sample knocking down ANGPTL4 was reduced, indicating that ANGPTL4 positively regulated the activation of EGFR. In the sample knocking down ANGPTL4, the expression of Akt and p-Akt (phosphorylated Akt) was reduced, indicating that ANGPTL4 positively regulated the activation of Akt. The expression of mTOR and p-mTOR (phosphorylated mTOR) in the sample knocking down ANGPTL4 was also reduced, indicating that ANGPTL4 positively regulated the activation of mTOR. The expression of EPB41L1, HER2, p-HER2 and other proteins was also reduced after knocking down ANGPTL4.

[0116] These results indicate that, like EPB41L1, ANGPTL4 can influence the activation of the EGFR signaling pathway, and key proteins within the pathway, such as phosphorylated EGFR (p-EGFR) and phosphorylated mTOR (p-mTOR), are significantly positively regulated by ANGPTL4. Therefore, Western blot results show that in samples with ANGPTL4 knockdown, the expression levels of p-EGFR and p-mTOR are reduced, suggesting that ANGPTL4 influences cellular behavior by enhancing the activation of the EGFR signaling pathway.

[0117] Finally, cell IC was used. 50 Measurement and colony formation assays further validated the effect of ANGPTL4 expression status on the sensitivity of lung adenocarcinoma cells to osimertinib treatment.

[0118] Detailed experimental methods and procedures are as described above, see the appendix of reference [4] (Supplementary Materials and Methods, page 7, line 8, 10. Colony formation assay), and experimental results are as follows. Figure 16-17 First, let's start with... Figure 16 In the clonogenic assay, different concentrations of osimertinib (1 μM, 2 μM, 5 μM) and ANGPTL4 gene knockdown (ANGPTL4_kd) cells were used to evaluate the clonogenic ability of tumor cells under different treatment conditions. The negative control group (DMSO) showed the clonogenicity of untreated tumor cells. The results showed that the number of colonies gradually decreased with increasing osimertinib concentration, indicating that osimertinib can effectively inhibit the clonogenic ability of cells. In ANGPTL4 gene knockdown cells, almost no clonogenicity was observed, indicating that ANGPTL4 knockdown can enhance the sensitivity of cells to osimertinib.

[0119] Then through Figure 17 Analysis of cell line IC 50 The results were used to assess the sensitivity of cells to osimertinib. The results showed that the IC50 of ANGPTL4 knockdown cells (ANGPTL4_kd) was significantly lower. 50 The value was significantly lower than that of the negative control group (2.33 μM vs. 5.51 μM, and the "**" in the figure indicates a statistically significant difference (p<0.01)), which indicates that knocking down ANGPTL4 can increase the sensitivity of cells to osimertinib.

[0120] In summary, through the analysis of cell line IC 50The determination and colony formation experiment confirmed that the expression state of ANGPTL4 can affect the sensitivity of lung adenocarcinoma cells to osimertinib treatment. Specifically, high expression of ANGPTL4 can lead to drug resistance of lung adenocarcinoma cells to osimertinib. The colony formation experiment directly shows that knocking down ANGPTL4 can enhance the sensitivity of cells to osimertinib, which is manifested as a significant decrease in the number of colony formation. 50 The determination further confirmed this, and the sensitivity of ANGPTL4 gene-knocked-down cells to osimertinib was significantly higher than that of the control group, which was manifested as a lower IC 50 value, that is, a low drug dose can achieve the same therapeutic effect as a high dose.

[0121] Example 7 Research on the mechanism of ANGPTL4 and EPB41L1 co-mediated EGFR-TKI drug resistance

[0122] 1. MDM2 / eIF5B / PD-L1 axis regulates secreted protein ANGPTL4 to mediate lung adenocarcinoma targeted therapy drug resistance

[0123] In view of the above examples to confirm that EPB41L1 and ANGPTL4 are regulated by nuclear PD-L1, and both are positively correlated with osimertinib resistance. At the same time, the ligand and receptor of membrane protein EPB41L1 and secreted protein ANGPTL4 are unknown, this embodiment will find the potential ligand-receptor relationship between EPB41L1 and ANGPTL4 and the binding domain existing between the proteins through molecular docking prediction.

[0124] The molecular docking prediction results are shown in the left side of Figure 18 , Figure 18 The left side of the figure shows the three-dimensional structure model of EPB41L1 (purple) and ANGPTL4 (yellow), as well as the simulation diagram of their interaction. The region of interaction between the two proteins is marked with a black line box, and the interaction of the amino acid residues in this region is shown in detail in the enlarged view below, including hydrogen bonds (represented by dashed lines) and other types of intermolecular interactions. The right side of the figure shows a column chart representing the contribution of different amino acid residues in the interaction between EPB41L1 and ANGPTL4, where certain residues have a higher contribution (such as 15.4%), which means that these residues play a key role in the binding of the two proteins. The sequence alignment diagram below shows the amino acid sequences of EPB41L1 and ANGPTL4 in the interaction region, where identical or similar amino acids are connected by dots or lines, indicating that these regions have structural and functional similarities.

[0125] 2. Verification of ANGPTL4 and EPB41L1 protein interaction in tumor cells

[0126] The above findings indicate that EPB41L1 and ANGPTL4 are involved in the regulation of the formation of osimertinib resistance through direct intermolecular interaction. Through molecular docking analysis, the key amino acid residues between the two proteins were determined, which interacted through hydrogen bonds and other intermolecular forces to form a stable binding interface. This direct intermolecular interaction affects the functions of EPB41L1 and ANGPTL4, EPB41L1 as a cytoskeleton regulatory protein regulates the cytoskeleton, ANGPTL4 as angiopoietin-like protein 4 is involved in angiogenesis and metabolism, and their interaction affects tumor cell migration or microenvironment remodeling, thereby affecting the sensitivity of cells to osimertinib.

[0127] After obtaining the simulation results of molecular docking, the co-immunoprecipitation experiment (Co-IP) was used to verify whether there was indeed a combination between EPB41L1 and ANGPTL4. The experimental method is described in the appendix of reference [4] (Materials and Methods, page 12, line 15, 20. Immunoprecipitation (IP) and Co-immunoprecipitation (Co-IP)) section.

[0128] The results are shown in Figure 19 The input in the co-immunoprecipitation experiment is the cell lysate as a control, showing the expression level of EPB41L1, ANGPTL4, EGFR and HER2 in total protein before immunoprecipitation, to ensure that these proteins are present at the beginning of the experiment. IgG is a negative control, and non-specific IgG antibody is used for immunoprecipitation. The results show that IgG does not specifically precipitate EPB41L1 or ANGPTL4, indicating that the precipitation in the experiment is specific. Immunoprecipitation using EPB41L1 antibody shows that ANGPTL4 is precipitated in addition to EPB41L1 itself, indicating that there is a direct interaction between EPB41L1 and ANGPTL4. Then, immunoprecipitation using ANGPTL4 antibody shows that EPB41L1 is precipitated in addition to ANGPTL4 itself, again supporting the hypothesis that there is a direct interaction between EPB41L1 and ANGPTL4. EGFR and HER proteins are also used in the experiment as a comparison, and the results show that they are also specifically precipitated by EPB41L1 and ANGPTL4, suggesting that ANGPTL4 and EPB41L1 have a close relationship with the ErbB family of proteins (also known as the HER family or the EGFR family).

[0129] Next, we tagged EPB41L1 with hemagglutinin (HA) tag and tagged ANGPTL4 with Flag tag, and then confirmed the interaction between EPB41L1 and ANGPTL4 again by repeated co-immunoprecipitation (Co-IP) with tag proteins. The experimental method is also referred to the annex of the literature [4] (Supplementary Materials and Methods, page 12, line 15, 20. Immunoprecipitation (IP) and Co-immunoprecipitation (Co-IP)) section.

[0130] Figure 20 The results show that when immunoprecipitation is performed using HA antibody, in addition to the HA-tagged EPB41L1 itself, the Flag-tagged ANGPTL4 is also precipitated. Similarly, when immunoprecipitation is performed using Flag antibody, in addition to the Flag-tagged ANGPTL4 itself, the HA-tagged EPB41L1 is also precipitated. At the same time, the negative control (IgG) in the experiment shows no non-specific precipitation, and the results further support that there is indeed a direct interaction between EPB41L1 and ANGPTL4, and this interaction plays an important biological function in cells, such as participating in signal transduction, regulating gene expression, or affecting cell behavior.

[0131] In summary, the results of the two Co-IP experiments show that when immunoprecipitation is performed using EPB41L1 antibody, ANGPTL4 protein is co-precipitated, and vice versa, which indicates that there is a direct interaction between EPB41L1 and ANGPTL4.

[0132] Example 8: Investigation of the interaction between EGFR, HER2, EPB41L1 and ANGPTL4

[0133] The above Example 7 verifies that there is a direct interaction between EPB41L1 and ANGPTL4. This example further explores the influence of EPB41L1 and ANGPTL4 on the EGFR signaling pathway and the ErbB signaling pathway, as well as the specific mechanism of action between EPB41L1 and ANGPTL4 and EGFR and HER2.

[0134] Investigation of the mechanism by which EPB41L1 affects the EGFR and ErbB signaling pathways

[0135] 1. Proteinomics sequencing (Shanghai iProteome Biotechnology Co., Ltd.) was used for further analysis, and the data found that EPB41L1 was closely related to the EGFR signaling pathway and the ErbB signaling pathway, especially the HER2 molecule (results are shown in Figure 21 ). Figure 21The left lower panel is a heat map of EPB41L1 expression level. The expression of EPB41L1 increases gradually from left to right in the tissues, and the protein heat map associated with the expression is shown. The right upper panel is the result of enrichment analysis, which lists the biological processes or signaling pathways significantly associated with EPB41L1 expression. The color bar indicates the range of P value, and the redder the color, the smaller the P value, and the more significant the correlation.

[0136] The right lower panel shows the relationship between EPB41L1 and other proteins. HER2 and PCNA (proliferating cell nuclear antigen) are labeled in the figure, indicating that EPB41L1 is closely related to these two molecules. The finding that EPB41L1 is closely related to HER2 molecules is confirmed by enrichment analysis, in which EGFR and ErbB signaling pathways show significant enrichment in biological processes related to EPB41L1 (P value less than 0.05). Figure 21 The direct relationship between EPB41L1 and HER2 is revealed, which indicates that EPB41L1 plays an important role in regulating HER2-related signaling.

[0137] Subsequently, western-blot experiments were performed to verify whether EPB41L1 or ANGPTL4 can inhibit EGFR and HER2 activation after knocking down EPB41L1 or ANGPTL4 genes. Figure 22 and 23 The experimental results show that knocking down EPB41L1 or ANGPTL4 can significantly inhibit the activation of EGFR and HER2, and the expression level of related proteins is significantly reduced. Specifically, the expression levels of p-HER2, p-MAPK and p-STAT3 are reduced after knocking down EPB41L1 or ANGPTL4, indicating that these two genes affect cell behavior by regulating the activation of HER2, MAPK and STAT3 signaling pathways. The results of this part of the experiment show that EPB41L1 and ANGPTL4 can affect cell behavior by affecting the activity of the HER2 signaling pathway, and the mechanism is discussed as follows.

[0138] 2. The direct binding between EGFR, HER2, ANGPTL4 and EPB41L1 was confirmed by GST-pulldown experiment.

[0139] See ([5] Luo YY, Ruan CS, Zhao FZ, Yang M, Cui W, Cheng X, Luo XH, Zhang XX, Zhang C. ZBED3 exacerbates hyperglycemia by promoting hepatic gluconeogenesis through CREB signaling. Metabolism. 2025 Jan; 162: 156049.) for specific experimental procedures:

[0140] 1) Construction of the target proteins with -GST and -His tags: Isopropyl-β-D- thiogalactoside (IPTG) was used to induce the expression of fusion proteins from pET28a-b-His and pGEX-4T-1-a vectors. When detecting the direct interaction between EPB41L1 or ANGPTL4 and EGFR or HER2, -GST tags were used to label EGFR and HER2 proteins, and His tags were used to label EPB41L1 and ANGPTL4 proteins. When detecting the direct interaction between EPB41L1 and ANGPTL4, -GST tags were used to label ANGPTL4, and His tags were used to label EPB41L1.

[0141] 2) Cell disruption and protein purification: Ultrasonic disruption, GST column affinity purification, and Ni column affinity purification were used to obtain purified target proteins, which were analyzed by SDS-PAGE and western blot.

[0142] See Figure 24-26 for experimental results. Among them, Figure 24 shows the results of protein purification. The left panel of the SDS-PAGE electrophoresis shows the purification results of GST-ANGPTL4. The arrow indicates the position of the target protein, which is approximately 55 kDa, which is basically consistent with the expected size of the ANGPTL4 fusion protein. The right panel shows the purification results of multiple GST and His fusion proteins (including EPB41L1-His, ANGPTL4-His, HER2, and EGFR). The arrow indicates the position of each target protein, confirming the successful expression and purification of these proteins. Figure 25The results of direct protein binding experiments are shown to detect the interaction between ANGPTL4 and EPB41L1. GST pulldown experiments were performed using the GST Protein Interaction Pull-Down Kit, and it can be seen that when GST-ANGPTL4 was co-incubated with EPB41L1-His, EPB41L1-His was successfully pulled down, indicating a direct interaction between the two. In contrast, no pull-down of EPB41L1-His was observed when using the GST-vector control, confirming that the interaction is specific. Figure 26 Further direct binding interactions between EGFR, HER2, ANGPTL4 and EPB41L1 were demonstrated. In the GST pulldown section, when GST-HER2 was co-incubated with EPB41L1-His or ANGPTL4-His, both were successfully pulled down, indicating a direct interaction with HER2. Similarly, when GST-EGFR was co-incubated with EPB41L1-His or ANGPTL4-His, both were also successfully pulled down, indicating a direct interaction with EGFR and HER2. These results indicate a complex interaction between EGFR, HER2, ANGPTL4 and EPB41L1.

[0143] 3. The binding relationship between the above proteins was also corroborated by molecular docking, which also found that EPB41L1 and ANGPTL4 have relevant domains for protein interaction with HER2 protein. Specifically as Figure 27 shown, the top left panel shows a three-dimensional structural model of EPB41L1 (orange) and HER2 (purple). The region of interaction between the two proteins is indicated by the dashed box, indicating a direct binding interface between EPB41L1 and HER2. The top right panel shows a detailed structure of the binding region between EPB41L1 and HER2, including possible hydrogen bonds (indicated by dashed lines) and other types of intermolecular interactions. The bottom left panel shows a three-dimensional structural model of ANGPTL4 (blue) and HER2 (purple). Again, the region of interaction between the two proteins is indicated by the dashed box, indicating a direct binding interface between ANGPTL4 and HER2. The bottom right panel shows a detailed structure of the binding region between ANGPTL4 and HER2, including possible hydrogen bonds and other intermolecular interactions.

[0144] Molecular docking results showed that both EPB41L1 and ANGPTL4 could form stable binding interface with HER2, which was achieved by forming hydrogen bonds and other intermolecular interactions. These findings supported the direct binding observed by GST-pulldown experiments and further revealed the interaction mechanism between these proteins.

[0145] Example 9 Screening of compounds targeting the membrane protein complex to reverse osimertinib resistance

[0146] 1. To investigate the binding mechanism and process between EGFR, HER2, ANGPTL4 and EPB41L1

[0147] According to the experimental results of Example 8, it is known that EGFR, HER2, ANGPTL4 and EPB41L1 can directly bind to each other, and EPB41L1 and ANGPTL4 have related domains that can interact with HER2 protein. Therefore, it is necessary to further understand how these four proteins bind to each other.

[0148] One possible way is that ANGPTL4 first interacts with EPB41L1, providing an opportunity for the further binding of EGFR and HER2. ANGPTL4 and EPB41L1 interact first to form a microenvironment on the cell membrane that is conducive to the binding of EGFR and HER2, thereby promoting the formation of EGFR / HER2 complex. The formation of EGFR / HER2 complex leads to the activation of downstream signaling pathways, including cell survival, proliferation and metastasis-related signaling pathways. Activated signaling pathways can help tumor cells adapt to the stress of EGFR-TKI treatment, leading to tumor resistance to EGFR-TKI. Figure 28 A schematic diagram showing the principle of the formation of the membrane protein complex is shown, Figure 28The nucleus PD-L1 regulates the expression of EPB41L1 and ANGPTL4, and the two form a complex, which further promotes the formation of EGFR / HER2 dimers, and the four form a new membrane protein complex. EPB41L1 and ANGPTL4 interact in the cytoplasm, which provides a platform or opportunity for the binding of EGFR and HER2. EGFR and HER2 can further form dimers on the cell membrane, and the formation of such dimers is affected by the interaction of EPB41L1 and ANGPTL4. Finally, the EGFR / HER2 signaling pathway mediates EGFR-TKI resistance. EGFR-TKI is a targeted therapeutic drug for EGFR mutations, but tumor cells activate the EGFR / HER2 complex and downstream signaling pathways through the above mechanism to obtain resistance to EGFR-TKI. In addition, since EGFR-TKI treatment is usually targeted at specific EGFR mutations, the formation of the EGFR / HER2 complex provides a mechanism to bypass these mutations, allowing tumor cells to continue to survive and proliferate.

[0149] Based on the speculation of the four protein binding processes, virtual screening of small molecule drugs was performed on the L4000 compound library to find compounds that can competitively bind EPB41L1 and ANGPTL4, and see if they can reduce the binding between EGFR and HER2 by competing with EPB41L1 or ANGPTL4. A group of small molecule compounds represented by SIYRY acetate (with the strongest affinity) was found. These compounds all inhibit the interaction between EPB41L1 and ANGPTL4 by competitively binding to the interaction interface between them. The chemical molecular structure of SIYRY acetate is as follows:

[0150]

[0151] The experimental results are shown in Figure 29wherein the left panel is a three-dimensional structural model of EPB41L1 and ANGPTL4, and the binding interface between them. The central region of the binding interface is represented by gray spheres, which is the site where small molecule compounds can intervene. The right panel is an enlarged view of the docking results of SIYRY acetate with the binding interface of EPB41L1 and ANGPTL4. The figure shows in detail the interactions between SIYRY acetate and the key amino acid residues of EPB41L1 and ANGPTL4, including hydrogen bonds (green dashed lines) and other types of intermolecular interactions. The figure further enlarges the details of the interactions of SIYRY acetate with the binding interface of EPB41L1 and ANGPTL4, showing how SIYRY acetate inhibits the binding of EPB41L1 and ANGPTL4 by forming hydrogen bonds with specific amino acid residues. The screening results show that SIYRY acetate performs best in terms of affinity, indicating that it is an effective inhibitor of the interaction between EPB41L1 and ANGPTL4. This inhibitory effect is achieved by blocking the direct binding between EPB41L1 and ANGPTL4, which in turn affects the activation of the EGFR / HER2 signaling pathway, ultimately affecting the proliferation, survival, and drug resistance of tumor cells.

[0152] To verify whether SIYRY acetate has the effect of blocking the interaction between EPB41L1 and ANGPTL4, a fluorescence resonance energy transfer (FRET) experiment was performed.

[0153] The specific steps of the FRET experiment are as follows: 1) Enzyme marker preliminary screening: the CFP-YFP double positive cells obtained by screening (see Wang Y, Zhang B, Zhang R, Ding D, Ma W, Wang W, Liu Z, Zhu Y, Wang X, Zhi D, Wang D. EGFP / RFP-based FRET sensors for botulinum neurotoxin A biological activity detection and methodological validation. Anal Chim Acta. 2025 Feb 1;1337:343546.) are plated in a 96-well plate, 4000 cells per well. Use complete culture medium (DMEM + 10% fetal bovine serum) to culture overnight to ensure adhesion. A total of 56 wells are set up, of which 53 are drug treatment groups to be screened and 3 are control groups. Dilute the candidate small molecule 1:1000 in DMEM complete culture medium and add it to the corresponding cell wells of the 96-well plate for 12 hours (h) of treatment. Turn on the enzyme marker and select the TR fluorescence mode, with excitation light at 515 nm and emission light at 480 nm. Detect the effect of different drugs on the interaction of the two proteins. To prevent the effect of drugs on cell number, use the BCA method for protein quantification to eliminate errors. Discard the culture medium and wash three times with PBS, then lyse the cells using RIPA lysis buffer and use the BCA method for protein quantification. After deducting the blank value, calculate the FRET / BCA value to screen out potential positive drugs. 2) Laser confocal secondary verification: take 6 35mm laser confocal imaging dishes, each containing 5000 cells, and use DMEM complete culture medium to culture for 24h. Dilute the candidate small molecule 1:1000 in DMEM complete culture medium and add it to the 35mm laser confocal imaging dishes, 3ml per dish. Continue to culture for 24h. Use an Olympus FV3000 laser confocal microscope, set the fluorescence detection channels at 488nm and 568nm, and use a 63x oil lens to take pictures. Use a circular tool to circle the light bleaching range (1um circle within the CFP and YFP overlap range), and use a 514nm laser to perform continuous light bleaching for 1s. Set 100s continuous measurement, then record the fluorescence intensity of CFP and YFP every 20s, and take pictures of the cells before and after light bleaching. Finally, detect the percentage increase in CFP fluorescence and calculate the interaction force of the two.

[0154] The experimental results confirm that the small molecule peptide SIYRY acetate has the strongest ability to inhibit the binding of ANGPTL4 and EPB41L1 Figure 30Aand B). Where Ctrl (control) is the energy transfer between ANGPTL4 (labeled as Protein A) and EPB41L1 (labeled as Protein B) without adding any inhibitors. The change of fluorescence intensity is also shown in the figure, where the red curve represents Protein A-CFP (donor) and the blue curve represents Protein B-YFP (acceptor). When the two bind, energy transfer occurs, resulting in a decrease in donor fluorescence intensity and an increase in acceptor fluorescence intensity. The ratio of ΔFluorescence CFP / YFP in the figure reflects the degree of energy transfer.

[0155] Specifically, Figure 30A It is shown that after adding SIYRY acetate, the energy transfer between ANGPTL4 and EPB41L1 is significantly reduced, and the energy transfer efficiency from the donor fluorescent protein ANGPTL4 (CFP, i.e. green fluorescent protein) to the acceptor fluorescent protein EPB41L1 (YFP, i.e. yellow fluorescent protein) is only 6.68%. This indicates that under experimental conditions, 6.68% of the fluorescent energy emitted by the donor fluorescent protein (CFP) is transferred to the acceptor fluorescent protein (YFP). This indicates a decrease in the efficiency of FRET, indicating an increase in the distance between the donor and acceptor and a decrease in interaction, which is direct evidence that SIYRY acetate effectively inhibits the binding of the two. In contrast, another compound, HAECGFT acetate, has a less significant effect on the binding of ANGPTL1 and EPB41L1 than SIYRY acetate. Figure 30B is the change in energy transfer after adding non-specific peptides (Nonapeptide) and Angiotensin III (as a control). It is shown that Substance P has a less significant effect on the binding of ANGPTL4 and EPB41L1 than SIYRY acetate.

[0156] The results of the FRET experiment show that SIYRY acetate significantly reduces the energy transfer between ANGPTL4 and EPB41L1, indicating that it can effectively inhibit the binding of the two. In contrast, other compounds such as HAECGFT acetate and Substance P also show some inhibitory effect, but the effect is not as significant as SIYRY acetate. These results indicate that SIYRY acetate is an effective inhibitor that can block the interaction between ANGPTL4 and EPB41L1, thereby affecting related signaling pathways and cell behavior.

[0157] Subsequently, the inhibitory effect of SIYRY acetate was verified again by changes in the expression levels of related proteins.

[0158] Co-IP experiments, the specific experimental method is the same as above, from Figure 31 From the upper panel of the results, after treatment with Pertuzumab (an inhibitor of EGFR / HER2 interaction) (1 mg / mL), it cannot block the binding of EPB41L1 to EGFR and HER2 proteins. However, when SIRYR acetate (10 μM) is added, the interaction between HER2 and EGFR and EPB41L1 is significantly weakened, indicating that SIRYR acetate blocks the binding of EPB41L1 to EGFR and HER2, and the latter two lose the pull-in and potential combination effect in the spatial position. Figure 31 The lower panel of FIG. 1 also shows similar effects. After protein extraction, SIRYR acetate treatment is added, and the interaction between EPB41L1 and HER2 and EGFR is also significantly reduced after the addition of SIRYR acetate, which further supports that SIRYR acetate can block the interaction between these proteins.

[0159] Further from the IP experiment results of Figure 32 , after overexpression of PD-L1, the interaction between EPB41L1, EGFR and HER2 is significantly enhanced. When SIRYR acetate is added, the binding ability between the above proteins is significantly weakened, further supporting that SIRYR acetate can inhibit the formation of EGFR / HER2 dimers by affecting the binding between target proteins, and thus inhibit the function of the corresponding signaling pathway.

[0160] In summary, through Co-IP experiments, it is observed that after treatment of cells with SIRYR acetate, the pairwise interaction between EGFR / HER2 / EPB41L1 is significantly blocked. In particular, the depolymerization of EGFR / HER2 dimers, this finding suggests that SIRYR acetate has the potential to reverse EGFR-TKI resistance. EGFR interacts with HER2 to form heterodimers, mediating EGFR-TKI resistance, and SIRYR acetate breaks the EGFR / HER2 binding by inhibiting the binding between ANGPTL4 and EPB41L1, which helps to restore the sensitivity of EGFR-TKI. It is also proved that reducing the binding force between ANGPTL4 and EPB41L1 is the core mechanism to solve the occurrence and development of EGFR-TKI resistance.

[0161] Example 10 Verification of SIRYR acetate targeting a novel protein complex to reverse osimertinib resistance

[0162] Based on Example 7, which demonstrated through changes in protein expression levels and in vitro cell experiments that inhibiting the binding between ANGPTL4 and EPB41L1 can reduce the formation of EGFR / HER2 dimers and decrease the occurrence of EGFR-TKI resistance, this application will verify this key finding.

[0163] By simultaneously knocking down PD-L1 in ARID1A-knockdown lung adenocarcinoma (HCC4006) cell lines, the role of PD-L1 in ARID1A-mediated EGFR-TKI resistance was reaffirmed. The experimental method was the same as above, and please refer to the appendix of reference [4] (Supplementary Materials and Methods, page 5, line 26, 7. Culture and stable transduction of cell lines and page 6, line 22, 8. Generating LUAD cell lines with integrated tet-inducible PTEN expression).

[0164] Western blot results showed ( Figure 33 The expression of several key proteins under different treatment conditions (control group, sh-PDL11, sh-PDL12, sh-PDL13, and sh-ARID1A) was investigated. In the sh-ARID1A treatment group, ARID1A expression was significantly reduced, validating the success of ARID1A knockdown. Simultaneously, PD-L1 expression was significantly increased after ARID1A knockdown, indicating that ARID1A plays a reverse regulatory role in PD-L1 expression. In the sh-PDL11-3 treatment group, PD-L1 expression was significantly reduced. After PD-L1 knockdown, p-EGFR expression was significantly reduced, indicating that PD-L1 participates in EGFR-TKI resistance by affecting EGFR phosphorylation. The experimental results, demonstrating that simultaneous PD-L1 knockdown in ARID1A-knockdown lung adenocarcinoma cell lines inhibited the above-mentioned ARID1A regulatory effects, further confirm the crucial role of PD-L1 in ARID1A-mediated EGFR-TKI resistance. ARID1A influences EGFR phosphorylation by regulating PD-L1 expression, thereby participating in the formation of EGFR-TKI resistance. Knockdown of PD-L1 significantly reduced p-EGFR expression, indicating that PD-L1 plays a crucial role in EGFR-TKI resistance. These findings provide new targets and strategies for overcoming EGFR-TKI resistance.

[0165] Then the binding site of SIRYR acetate in EGFR / HER2 / EPB41L1 / ANGPTL4 protein complex was predicted by molecular docking, and the position of SIRYR acetate in the complex was marked with red in the figure, showing that it was inlaid in the central part of the complex. Figure 34 The binding site of SIRYR acetate in EGFR / HER2 / EPB41L1 / ANGPTL4 protein complex was successfully predicted by molecular docking. SIRYR acetate can be inlaid in the central part of the complex, which means that SIRYR acetate can directly affect the interaction between these proteins, thereby interfering with their functions.

[0166] It is confirmed by the above experimental results that ANGPTL4 as a secreted protein is completed by acting on the EGFR / HER2 / EPB41L1 complex to complete the ligand binding, and then plays a role in regulating the downstream. In order to verify the ligand effect of ANGPTL4, the present application uses recombinant ANGPTL4 protein to treat ANGPTL4 knockdown cells, and finds that it can compensate for the inhibition of gene knockdown and reactivates the EGFR signaling pathway. Figure 35 The western-blot experiment shows the expression of several key proteins under different treatment conditions (control group, sh-ANGPTL4, ANGPTL4 protein treatment group). In the sh-ANGPTL4 treatment group, the expression of ANGPTL4 is significantly reduced, verifying the success of ANGPTL4 knockdown. In the treatment group supplemented with ANGPTL4 protein, the expression of ANGPTL4 is restored, indicating that the recombinant ANGPTL4 protein successfully supplements the function of the knocked-down ANGPTL4.

[0167] In the sh-ANGPTL4 treatment group, the expression of p-EGFR is significantly reduced, indicating that the EGFR signaling pathway is inhibited. In the treatment group supplemented with ANGPTL4 protein, the expression of p-EGFR is restored, indicating that the recombinant ANGPTL4 protein reactivates the EGFR signaling pathway. Again, it is proved that ANGPTL4 plays a key role in regulating the EGFR signaling pathway, and its overexpression can stimulate the EGFR signaling pathway to be more active.

[0168] Figure 36-37 The western-blot is in the PD-L1 overexpression or ARID1A knockdown (both can induce EGFR-TKI resistance) cell line with the addition of SIRYR acetate treatment, and the results confirm that SIRYR acetate can significantly inhibit the EGFR signaling pathway and the expression of HER2 protein.

[0169] Protein expression analysis is as follows:

[0170] Figure 36 The expression of several key proteins under different treatment conditions (control group, PD-L1 overexpression group, SIRYR acetate treatment group) is shown. Among them, the expression of PD-L1 is significantly increased in the PD-L1 overexpression group. At the same time, the expression of p-EGFR and HER2 also increases, indicating that PD-L1 overexpression activates the EGFR signaling pathway and increases the expression of HER2.

[0171] After adding SIRYR acetate treatment, the expression of p-EGFR and HER2 is significantly reduced, indicating that SIRYR acetate inhibits the activation of the EGFR signaling pathway and the expression of HER2. At the same time, the expression of EPB41L1 is also reduced in the SIRYR acetate treatment group, further supporting the inhibitory effect of SIRYR acetate on the EGFR signaling pathway.

[0172] Figure 37 The effect of ARID1A knockdown on the expression of the above several proteins is analyzed. In the ARID1A knockdown group, the expression of ARID1A is significantly reduced, verifying the success of ARID1A knockdown.

[0173] At the same time, the expression of p-EGFR and HER2 also increases, indicating that ARID1A knockdown activates the EGFR signaling pathway and increases the expression of HER2.

[0174] In the SIRYR acetate treatment group, the expression of p-EGFR and HER2 is significantly reduced, indicating that SIRYR acetate inhibits the activation of the EGFR signaling pathway and the expression of HER2. The expression of EPB41L1 is also reduced in the SIRYR acetate treatment group, further supporting the inhibitory effect of SIRYR acetate on the EGFR signaling pathway.

[0175] In summary Figure 36 and 37The results found that in PD-L1 overexpression or ARID1A knockdown (both can induce EGFR-TKI resistance) cell lines, the addition of SIRYR acetate treatment can significantly inhibit the EGFR signaling pathway and HER2 protein expression. This finding indicates that SIRYR acetate overcomes EGFR-TKI resistance by inhibiting the EGFR signaling pathway. Specifically, after adding SIRYR acetate, the expression of p-EGFR and HER2 can be significantly reduced, thereby inhibiting the activation of the EGFR signaling pathway. In addition, SIRYR acetate can also reduce the expression of EPB41L1, further supporting its inhibitory effect on the EGFR signaling pathway.

[0176] Example 11 In vivo verification of SIRYR acetate targeting novel protein complex to reverse osimertinib resistance

[0177] The above examples have shown through protein expression levels and in vitro cell experiments that SIRYR acetate blocks the interaction between ANGPTL4 and EPB41L1, further disrupting the formation of EGFR / HER2 protein dimers, thereby reversing osimertinib resistance. This example continues to verify the above effects through in vivo experiments, reducing the interaction between ANGPTL4 and EPB41L1 through the competitive binding effect of SIRYR acetate, and affecting the phenomenon of osimertinib resistance.

[0178] The experimental design is as follows: different treatment groups were used in the experiment, including the control group (DMSO), the osimertinib alone treatment group, the SIRYR acetate alone treatment group, and the combination treatment group.

[0179] The specific operation method of the animal experiment can be completed by referring to the appendix of the literature [4] (supplementary materials and methods, page 15, line 4, 26. In vivo xenograft model). Figure 38 The left graph shows the tumor samples of different treatment groups at the end of the experiment, which intuitively shows the difference in tumor volume. It can be seen that the tumor samples of the combination treatment group are significantly smaller than those of the other groups, further supporting the data in the graph.

[0180] Figure 38 The right graph shows the relative change in tumor volume of different treatment groups within 25 days.

[0181] The tumor volume of the sh-Ctrl:DMSO (green line) and sh-ARID1A:DMSO (black line) groups increased significantly, indicating that the tumor grew rapidly without drug intervention.

[0182] sh-Ctrl:Osimertinib (orange line) and sh-ARID1A:Osimertinib (blue line) groups, indicating that osimertinib has an effect of inhibiting tumor growth on the control group, and the tumor of the sh-ARID1A group shows the characteristics of osimertinib resistance.

[0183] The tumor volume of the sh-ARID1A:SIRYR acetate (red line) group also grows slowly, and SIRYR acetate alone cannot effectively control the tumor.

[0184] The tumor volume of the sh-ARID1A:Osimertinib+SIRYR acetate (purple line) group grows the slowest, indicating that the combination therapy significantly reverses the EGFR-TKI resistance characteristics.

[0185] Summary

[0186] In vivo experiments verified that osimertinib combined with SIRYR acetate treatment can significantly enhance the efficacy of EGFR-TKI. Specifically, using osimertinib or SIRYR acetate alone can inhibit tumor growth to some extent, but when used in combination, the inhibitory effect on tumor growth is the most significant. Specifically, the tumor volume of the combination therapy group grows the slowest, and even shows a trend of tumor volume reduction in the later stage of the experiment. This indicates that SIRYR acetate enhances the anti-tumor activity of osimertinib through a competitive mechanism, thereby overcoming EGFR-TKI resistance.

[0187] Summary:

[0188] The research process of the above technical solutions is summarized in the overall flowchart Figure 39 . In general, in cell experiments and in vivo experiments of PD-L1 overexpression or ARID1A knockdown (both of which can induce EGFR-TKI resistance), it is found that SIRYR acetate can significantly reverse the characteristics of EGFR-TKI resistance. Mechanistically, SIRYR acetate can effectively inhibit the interaction between ANGPTL4 and EPB41L1, thereby blocking the formation of EGFR / HER2 dimers, so that the downstream signaling pathway cannot be continuously activated, and this process is the key to reversing EGFR-TKI resistance. The discovery of this mechanism opens up a new treatment approach to tackle the problem of EGFR-TKI resistance. For example, EGFR-TKI is combined with small molecule compounds that competitively bind to ANGPTL4 / EPB41L1 for combination therapy, or related antibodies are combined into a complex preparation for administration. This combination therapy strategy provides a new treatment option for clinical treatment of EGFR-TKI-resistant non-small cell lung cancer patients.

[0189] The above description of the embodiments is intended to aid the ordinary skilled person in understanding and using the present application. The skilled person can easily make various modifications to the embodiments and apply the general principles described in the description to other examples without inventive effort. The present application is therefore not limited to these embodiments, and any improvements and modifications made on the basis of the disclosure of the present application shall fall within the scope of the present application.

Claims

1. The use of a compound in the preparation of a drug for inhibiting or reversing resistance to epidermal growth factor receptor tyrosine kinase inhibitors, characterized in that, The compound has the following molecular structural formula:

2. The application according to claim 1, characterized in that, The drug inhibits or reverses resistance to the epidermal growth factor receptor tyrosine kinase inhibitor by inhibiting the interaction between ANGPTL4 protein and EPB41L1 protein, thereby blocking the formation of EGFR / HER2 heterodimer.

3. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable additives or excipients.

4. A method for screening potential drugs to inhibit or reverse resistance to epidermal growth factor receptor tyrosine kinase inhibitors, characterized in that, include: The inhibitory effect of the candidate drug on the binding of ANGPTL4 protein to EPB41L1 protein was tested.

5. The method according to claim 4, characterized in that, Also includes: The inhibitor was mixed in vitro with a system containing the ANGPTL4 protein and the EPB41L1 protein.

6. The method according to claim 4, characterized in that, The testing of the candidate drug's inhibitory effect on the binding of ANGPTL4 and EPB41L1 proteins included: The candidate drug was co-incubated with the ANGPTL4 protein and the EPB41L1 protein, and the inhibitory effect of the candidate drug on the binding of the ANGPTL4 protein and the EPB41L1 protein was detected.

7. A kit for detecting whether a patient is resistant to an epidermal growth factor receptor tyrosine kinase inhibitor, characterized in that, The kit includes reagents for detecting the binding levels of ANGPTL4 protein and EPB41L1 protein in the patient's test sample, wherein the binding levels are positively correlated with drug resistance.

8. The reagent kit according to claim 7, characterized in that, Also includes: Standards used to detect the binding levels of the ANGPTL4 protein and the EPB41L1 protein.

9. The reagent kit according to claim 7, characterized in that, The kit is used to test human or animal subjects who have received treatment with growth factor receptor tyrosine kinase inhibitors.

10. A method for reducing or interfering with the resistance of epidermal growth factor receptor tyrosine kinase inhibitors in in vitro cell lines, characterized in that, include: The activation of downstream signaling pathways is inhibited by interfering with the binding of ANGPTL4 protein to EPB41L1 protein.

11. The method according to claim 10, characterized in that, The downstream signaling pathways include the EGFR and HER2 signaling pathways.

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