Application of YTHDF1 in regulation and control of tryptophan metabolism to promote formation of hepatocellular carcinoma immunosuppression microenvironment

Abstract

The invention discloses a key effect and a targeted intervention strategy of YTHDF1 in regulation and control of a hepatocellular carcinoma immunosuppression microenvironment. In the tumor-associated fibroblasts, YTHDF1 enhances the translation efficiency of OTUD5 mRNA through an m6A dependency mechanism, an up-regulated deubiquitination enzyme OTUD5 is directly combined with a tryptophan metabolic rate-limiting enzyme IDO1, ubiquitination degradation of the tryptophan metabolic rate-limiting enzyme IDO1 is inhibited, tryptophan is further driven to catabolism to generate kynurenine, regulatory T cell differentiation is promoted, and an immunosuppression microenvironment is formed. Spatial transcriptomics shows that YTHDF1 + CAFs and Tregs are significantly co-localized in tumor tissues and have metabolic interaction. Based on artificial intelligence virtual screening, it is found that a natural compound hesperidin can be specifically combined with a YTH structural domain of YTHDF1, an OTUD5-IDO1 signal axis mediated by the natural compound hesperidin is blocked, and the KYN level is remarkably reduced. Animal experiments prove that the tumor inhibition rate of combined treatment of hesperidin and anti-PD-1 is increased by 40% or above compared with that of a single drug group, and synergistic interaction is achieved by inhibiting Tregs infiltration and enhancing the activity of cytotoxic T lymphocytes.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to the application of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma. Background Art

[0002] Tumors have become the second leading cause of death globally, and the incidence rate continues to rise, seriously threatening human health. During the occurrence and development of tumors, the tumor microenvironment (TME) plays a key regulatory role.

[0003] The TME is composed of tumor cells, stromal cells, immune cells, and extracellular matrix, etc., and forms a synergistic network through interactions such as metabolism, mechanical signal transduction, and immune regulation, showing significant spatiotemporal heterogeneity. The TME not only provides nutritional support for tumor cells but also promotes the malignant progression of tumors through abnormal angiogenesis and immune escape.

[0004] In China, the incidence and mortality rates of liver cancer rank first globally, and hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancer. Although certain progress has been made in the treatment of HCC in recent years, most patients still face challenges such as treatment resistance and disease progression. Therefore, achieving more precise and effective treatment remains the focus of clinical and basic research. With the in-depth study of HCC, researchers have gradually recognized the complexity of the TME and its important role in immunotherapy, and are committed to improving the treatment effect of HCC by remodeling the immunosuppressive microenvironment. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art, and to propose the application of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma.

[0006] To achieve the above purpose, the present invention adopts the following technical solution: the application of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma.

[0007] Further, the YTHDF1 regulates the HCC immune microenvironment.

[0008] Further, the YTHDF1 enhances tryptophan catabolism.

[0009] Further, the YTHDF1 promotes the translation of OTUD5 mRNA, and OTUD5 directly interacts with IDO1 and inhibits its ubiquitination and degradation.

[0010] Further, hesperidin improves the efficacy of anti-PD-1 treatment by targeting YTHDF1.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This study for the first time reveals the key mechanism by which YTHDF1 in cancer-associated fibroblasts (CAFs) regulates the OTUD5-IDO1 signaling axis through an m6A-dependent mechanism, drives the reprogramming of tryptophan metabolism, and promotes the formation of an immunosuppressive microenvironment, breaking through the limitations of single targeting of tumor cells or immune checkpoints in traditional liver cancer treatment; innovatively discovers that the natural compound hesperidin can specifically bind to the YTH domain of YTHDF1, effectively block the immunosuppressive metabolic pathway mediated by it, and achieve synergistic enhancement through combination with anti-PD-1 therapy (the tumor inhibition rate is increased by more than 40%), providing a dual-target synergistic intervention strategy for liver cancer immunotherapy; in addition, the drug development mode based on artificial intelligence virtual screening and molecular dynamics verification significantly improves the discovery efficiency of targeted RNA epigenetic regulators, opening up a new way for the clinical transformation of the metabolism-immune cross-regulation network. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Experimental diagrams for clarifying the expression of YTHDF1 and its effect on the progression of HCC; Figure 2 Experimental diagrams for exploring the way by which YTHDF1 in CAFs regulates the HCC immune microenvironment and its specific molecular biological mechanism; Figure 3 Experimental diagrams for spatial transcriptomics to reveal the spatial interaction between YTHDF1+ CAFs and Tregs; Figure 4 Experimental diagrams for YTHDF1 promoting the production of KYN by enhancing TRP catabolism; Figure 5 Experimental diagrams for YTHDF1 promoting the translation of OTUD5 mRNA in an m6A-dependent manner; Figure 6 Experimental diagrams for OTUD5 directly binding to IDO1 and inhibiting its ubiquitin-proteasome degradation; Figure 7 Experimental diagrams for hesperidin significantly enhancing the efficacy of anti-PD-1 therapy by targeting YTHDF1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0013] To further understand the purpose, structure, characteristics, and functions of the present invention, the following is a detailed description in conjunction with the embodiments.

[0014] Example 1: Regulation of the HCC immune microenvironment by cancer-associated fibroblast YTHDF1.

[0015] RNA modification, as a frontier field of epigenetic research, plays a key role in post-transcriptional gene regulation.

[0016] The abundance differences of RNA modifications, including m1A, m3C, m5C, m6A, m7G, ac4C, m5U, and hm5C, were detected in 20 pairs of HCC tumor and adjacent tissues using LC-MS / MS to explore the role of RNA modifications in the TME.

[0017] The results showed that, as Figure 1 shown in A-1B, the m6A modification was significantly upregulated in tumor tissues. The m6A modification is a dynamic reversible process regulated by RNA modification proteins (RMPs). Therefore, it was speculated that the dysregulation of RMPs might lead to the elevated level of m6A modification in HCC tumor tissues.

[0018] Through bioinformatics analysis of large-sample HCC sequencing data, it was found that both YTHDF1 and RBMX showed significant abnormal expression, and the expression level of YTHDF1 was closely related to the patient survival period ( Figure 1 C).

[0019] By selecting YTHDF1 as the key gene for subsequent research. The TME consists of various cells such as tumor cells, stromal cells, and immune cells. Further single-cell RNA sequencing (scRNA-seq) data analysis showed that YTHDF1 was generally highly expressed in cancer-associated fibroblasts (CAFs) in HCC tumor tissues ( Figure 1 D).

[0020] As one of the significant features of the HCC microenvironment, CAFs regulate tumor progression through multiple mechanisms. To further explore the role of YTHDF1 in CAFs, an immunofluorescence (IF) experiment was conducted on the tumors and paired adjacent tissues of 100 HCC patients, and it was found that YTHDF1 was significantly upregulated in CAFs compared with normal tissues ( Figure 1 E).

[0021] Through multispectral immunohistochemistry (mIHC) analysis, it was found that a large number of YTHDF1+ CAFs infiltrated in the tumor tissues of advanced HCC patients, indicating a significant correlation between YTHDF1 and tumor progression ( Figure 1 F-1G).

[0022] The specific role of YTHDF1 in HCC progression was investigated by using fluorescence-activated cell sorting (FACS) technology to isolate different types of primary fibroblasts from the tumor tissues of patients and performing transcriptome sequencing on them.

[0023] Gene set enrichment analysis (GSEA) showed that YTHDF1 was significantly associated with immune-related signaling pathways, suggesting its important role in regulating immune responses ( Figure 1 H-1J).

[0024] Figure 1 Among them, (A) the overall experimental protocol of this study; (B) LC-MS / MS detection of the abundance differences of RNA modifications in 20 pairs of HCC tumor and adjacent tissues; (C) integrated analysis of three large-scale HCC sequencing datasets and combined with survival analysis to identify m6A regulatory factors abnormally expressed in HCC; (D) analysis of the mRNA expression levels of YTHDF1 in the main cell types of the TME using five single-cell RNA sequencing datasets; (E) representative images of IF staining of TMA and quantitative analysis; (F-G) representative images of mIHC staining of HCC tumor tissues at different stages and quantitative analysis, scale bar = 100 μm; (H) FACS strategy, sorting EpCAM−CD45−CD31−NG2−PDPN+ CAFs after excluding doublet cells and dead cells; (I) flow cytometry identification of YTHDF1low and YTHDF1high CAFs; (J) RNA-seq analysis of YTHDF1-related immune signaling pathways. ***p<0.001.

[0025] Example 2: YTHDF1 enhances tryptophan catabolism.

[0026] By constructing fibroblasts YTHDF1-specific knockout mice (Ythdf1F-KO) and establishing a primary HCC model, the specific role of YTHDF1 in CAFs was explored.

[0027] From Figure 2 A-2C, the number of tumors and the maximum tumor volume of Ythdf1F-KO mice were significantly lower than those of the control group.

[0028] From Figure 2 D-2F, in the mouse orthotopic HCC model, the deletion of YTHDF1 significantly inhibited tumor growth.

[0029] To further explore the role of YTHDF1 in CAFs, from Figure 2 G-2K, by using the 10X Genomics platform for single-cell RNA sequencing (scRNA-seq), it was found that the infiltration of immunosuppressive regulatory T cells (Tregs) in the tumor tissues of Ythdf1F-KO mice was significantly reduced, and the infiltration of CTLs increased. These results all indicate that YTHDF1 in CAFs plays an important role in Tregs infiltration.

[0030] Figure 2Among them, (A) Schematic diagram of the DEN / CCL4-induced primary HCC model in mice; (B) Evaluation of tumor growth ability by MRI and representative liver images from Ythdf1flox / flox and Ythdf1F-KO mice are shown; (C) Quantitative analysis of the number of tumors and the maximum tumor diameter in each group; (D) Schematic diagram of the mouse orthotopic HCC model constructed by intrahepatic injection of Hepa 1-6 cells; (E) Evaluation of tumor growth ability by small animal visible light and representative liver images from Ythdf1flox / flox and Ythdf1F-KO mice are shown; (F) Quantitative analysis of luciferase activity and tumor size in each group; (G) Schematic diagram of the scRNA-seq sample processing flow; (H) UMAP dimensionality reduction shows tumor-infiltrating CD45+ immune cells, and the density plot shows the distribution of cell populations; (I) Statistical chart of each immune cell type; (J) Flow cytometry experiments detect tumor-infiltrating Tregs (CD4+, CD25+, and FOXP3+) and CTLs (CD8+ and Granzyme B+) in Ythdf1flox / flox and Ythdf1F-KO mice; (K) Quantitative analysis of tumor-infiltrating Tregs and CTLs in Ythdf1flox / flox and Ythdf1F-KO mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0031] To further understand the spatial relationship between CAFs and Tregs in the TME, spatial transcriptome sequencing was performed on tumor tissues of HCC patients, and the tumor tissues were divided into multiple spatial domains by Visium spatial gene expression analysis. The analysis results showed that the YTHDF1+ CAF subset and Tregs were significantly co-localized spatially, and their interaction was closely related ( Figure 3 A-3D). Further research found that YTHDF1high CAFs and Tregs always maintained close contact, indicating an important spatial interaction between them ( Figure 3 B).

[0032] Figure 3Among them, (A) H&E staining images of tumor tissue sections from two HCC patients (P1 and P2); identifiable spatial domains were obtained by BASS analysis using spatial clustering method, further characterizing the spatial distribution characteristics of tumor cells, YTHDF1+ CAFs, and Tregs in tissue sections; the scatter plot shows the Spearman correlation analysis of the feature set scores of YTHDF1+ CAFs and Tregs; (B) spatial distribution of the distance between the nearest neighbor points between YTHDF1+ CAFs or YTHDF1- CAFs and Tregs in P1 and P2 tumor tissue sections; (C) representative images of mIHC staining of VIM, YTHDF1, CD4, and FOXP3 in HCC tumor tissue, scale bar = 50 μm; (D) analyzing the mIHC staining results to quantify the average distance of Tregs from the nearest YTHDF1low or YTHDF1high CAFs.

[0033] The specific mechanism of the interaction between YTHDF1high CAFs and Tregs was further investigated. CAFs and T cells were co-cultured under direct contact and non-contact conditions using a three-dimensional culture system and a two-chamber culture system.

[0034] The results showed that YTHDF1high CAFs could promote the differentiation of Tregs, and this effect was independent of direct contact ( Figure 4 A-4B).

[0035] After analyzing the conditioned medium of CAFs, it was found that the function of YTHDF1 mainly depended on metabolites rather than proteins ( Figure 4 C).

[0036] Metabolic reprogramming plays an important role in regulating the function of immune cells. This process is not limited to tumor cells but also involves complex metabolic interactions between stromal cells and immune cells. LC-MS / MS metabolomics analysis was performed on the conditioned medium of CAFs with high YTHDF1 expression, and it was found that the metabolites in the tryptophan (TRP) metabolic pathway changed most significantly ( Figure 4 D-4F).

[0037] TRP metabolism is closely related to immune escape, and kynurenine (KYN) is a key metabolite that promotes the differentiation of Tregs. Mass spectrometry analysis further confirmed that the upregulation of YTHDF1 enhanced the synthesis of KYN, and the KYN level was positively correlated with the expression of YTHDF1 in CAFs ( Figure 4 G-4K). These results indicate that YTHDF1 in CAFs promotes the production of KYN by enhancing tryptophan catabolism, thereby promoting the differentiation of Tregs and forming an immunosuppressive microenvironment ( Figure 4 L).

[0038] Figure 4 In (A - B), schematic diagram of the co - culture experimental design; Flow cytometry was used to detect the proportion of Tregs in CD4+ T cells in each group; (C) T cells were cultured alone, or T cells were cultured with the conditioned medium (CM) of YTHDF1high CAFs after different treatments, and flow cytometry was used to detect the proportion of Tregs; (D) Schematic diagram of the metabolomics experimental design; (E) Metabolic pathways enriched with metabolites showing significant changes in metabolomics pathway analysis; (F) Metabolite set enrichment analysis of tryptophan (TRP) metabolism between the two groups; (G) Correlation between YTHDF1 expression level and the KYN / TRP ratio in cell supernatants; (H) Ythdf1flox / flox and Ythdf1F - KO mouse HCC models were constructed, and the KYN / TRP ratio in mouse plasma was detected; (I) Isotope tracing experiment was carried out using 13C - labeled TRP (13C11 - TRP), and the production of 13C10 - KYN was quantitatively analyzed by HPLC - MS / MS; (J) Representative images of immunofluorescence (IF) staining of KYN in tumor tissues of HCC patients, scale bar = 100 μm; (K) Pearson correlation analysis was used to evaluate the correlation between KYN level in HCC patients and YTHDF1 expression level in CAFs; (L) Schematic diagram of the mechanism by which CAFs promote Tregs differentiation by secreting KYN. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant.

[0039] Example 3: YTHDF1 promotes the translation of OTUD5 mRNA, and OTUD5 directly interacts with IDO1 and inhibits its ubiquitination and degradation.

[0040] To reveal the molecular mechanism by which YTHDF1 in CAFs enhances tryptophan (TRP) catabolism, first, m6A - specific methylated RNA immunoprecipitation sequencing (MeRIP - seq) and RNA immunoprecipitation sequencing (RIP - seq) were used to identify the downstream targets of YTHDF1 ( Figure 5 A). Gene Ontology (GO) analysis showed that these target genes were significantly enriched in the process of protein deubiquitination ( Figure 5 B).

[0041] Through proteomic data analysis, OTUD1 and OTUD5 were screened as potential deubiquitinating enzymes regulated by YTHDF1 ( Figure 5 C).

[0042] Functional experimental results showed that OTUD5 was significantly correlated with kynurenine (KYN) production and Tregs differentiation, indicating that OTUD5 is a potential target of YTHDF1 ( Figure 5 D - 5E).

[0043] To further understand the molecular mechanism by which YTHDF1 regulates OTUD5, co-localization of YTHDF1 and OTUD5 mRNA was confirmed by 3D reconstruction fluorescence co-localization experiments ( Figure 5 F).

[0044] Subsequently, the m6A modification status of OTUD5 mRNA was confirmed by MeRIP-qPCR experiments, and the binding relationship between YTHDF1 and OTUD5 mRNA was demonstrated by RIP-qPCR ( Figure 5 G-5H).

[0045] Furthermore, point mutations were introduced into the key enzymatic sites of YTHDF1 (K395A and Y397A). The results showed that the binding of the mutated YTHDF1 to OTUD5 mRNA was significantly weakened, indicating that this binding is dependent on m6A modification ( Figure 5 I).

[0046] Polysome profiling further showed that knockdown of YTHDF1 led to a significant decrease in the polysome signal of OTUD5 mRNA and its transfer to the non-polysome fraction ( Figure 5 J), while upregulation of YTHDF1 had the opposite effect ( Figure 5 K). These results indicate that YTHDF1 promotes the translation of OTUD5 mRNA in an m6A-dependent manner.

[0047] Figure 5In (A), schematic diagram of screening downstream targets of YTHDF1 in CAFs using combined analysis of MeRIP-seq, RIP-seq, and proteomics; (B) GO analysis showed that potential YTHDF1-regulated targets were mainly enriched in the process of protein deubiquitination; (C) combined analysis of genes enriched in the process of protein deubiquitination and proteins upregulated with YTHDF1 overexpression in proteomics yielded candidate downstream molecules OTUD1 and OTUD5; (D) screening of candidate molecules by detecting the KYN / TRP ratio in cell supernatants; (E) screening of candidate molecules by detecting Tregs differentiation through co-culture experiments; (F) representative 3D reconstruction images of IF of YTHDF1 (green) and FISH of OTUD5 mRNA (red), showing the co-localization of YTHDF1 and OTUD5 mRNA in CAFs, scale bar = 20 μm; (G) MeRIP-qPCR verification of m6A modification of OTUD5 mRNA in CAFs; (H) YTHDF1 RIP-qPCR confirmation of the interaction between YTHDF1 and OTUD5 mRNA; (I) schematic diagram of wild-type (YTHDF1-WT) and mutant (YTHDF1-MUT) YTHDF1 constructs, Flag RIP-qPCR analysis showed the interaction between Flag-tagged YTHDF1-WT or YTHDF1-MUT and OTUD5 mRNA; (J-K) Polysome profiling analysis of the effect of YTHDF1 knockdown or overexpression on the translation level of OTUD5 mRNA. **p < 0.01, ***p < 0.001, n.s. = not significant.

[0048] Next, to further explore the mechanism by which YTHDF1 regulates KYN production through OTUD5, immunoprecipitation mass spectrometry (IP-MS) was used to screen for interacting proteins of OTUD5 and integrated analysis was performed with the TRP catabolism-related gene set. The results showed that IDO1, as the rate-limiting enzyme of TRP catabolism, might be a key downstream effector molecule of OTUD5 ( Figure 6 B). The interaction between OTUD5 and IDO1 in CAFs was further verified by co-immunoprecipitation experiment (Co-IP) ( Figure 6 C-6D). The direct interaction between OTUD5 and IDO1 was confirmed by GST pull-down assay and protein proximity ligation assay (PLA) ( Figure 6 E-6F). Further molecular docking analysis revealed the potential binding domain between OTUD5 and IDO1 ( Figure 6 G). Given that OTUD5 is a deubiquitinating enzyme, further investigation was carried out to determine whether it regulates the protein stability of IDO1.

[0049] The results showed that knockdown of OTUD5 significantly reduced the protein level of IDO1, while overexpression of OTUD5 had the opposite effect ( Figure 6 H).

[0050] In addition, under the treatment of the protein synthesis inhibitor cycloheximide (CHX), upregulation of OTUD5 could effectively maintain the stability of IDO1. The proteasome inhibitor MG132, rather than chloroquine (CQ), could reverse the decrease in IDO1 level caused by OTUD5 knockdown, indicating that OTUD5 might regulate the stability of IDO1 through the ubiquitin-proteasome pathway ( Figure 6 I). Further analysis found that overexpression of OTUD5 significantly reduced the ubiquitination level of IDO1, while knockdown of OTUD5 had the opposite effect ( Figure 6 J). Collectively, these results suggest that YTHDF1 promotes the translation of OTUD5 mRNA in an m6A-dependent manner, and OTUD5 binds to IDO1 and inhibits its ubiquitin-proteasome degradation, thereby enhancing TRP catabolism and promoting the formation of an immunosuppressive microenvironment ( Figure 6 K).

[0051] Figure 6 In (A), schematic diagram of OTUD5 IP experiment and subsequent LC-MS / MS analysis; (B) combined analysis of OTUD5 IP-MS interactomics (n = 334, unique peptides>5) and TRP catabolism-related gene set (n = 32); (C) exogenous Co-IP experiment to evaluate the interaction between OTUD5 and IDO1 after transfection of Flag-tagged OTUD5 and HA-tagged IDO1 plasmids in HEK293T cells; (D) endogenous Co-IP experiment to confirm the interaction between OTUD5 and IDO1 in CAFs; (E) GST pull-down experiment showing the direct binding of OTUD5 and IDO1, with purified GST as a control; (F) detection of the interaction between OTUD5 and IDO1 in CAFs by PLA experiment, scale bar = 5μm; (G) prediction of the interaction domains between OTUD5 and IDO1 by molecular docking; (H) WB experiment to detect the protein expression level of IDO1 in OTUD5 knockdown or overexpressed CAFs; (I) WB experiment to detect the change in IDO1 protein expression level in CAFs after CHX treatment for different times; WB experiment to detect the effect of OTUD5 knockdown on the protein expression level of IDO1 in CAFs after treatment with DMSO, MG132 or CQ respectively; (J) ubiquitination experiment to detect the ubiquitination level of IDO1 in OTUD5 knockdown or overexpressed CAFs; (K) Schematic diagram of the mechanism by which YTHDF1 promotes the translation of OTUD5 mRNA in an m6A-dependent manner, upregulated OTUD5 directly interacts with IDO1 and inhibits its ubiquitination degradation, thereby enhancing TRP catabolism and accelerating KYN production.

[0052] Example 4: Hesperidin enhances the efficacy of anti-PD-1 therapy by targeting YTHDF1.

[0053] To search for a pharmacological inhibitor specifically targeting YTHDF1, based on the crystal structure of the YTH domain of YTHDF1, artificial intelligence-assisted virtual screening was performed ( Figure 7 A).

[0054] Among 2,641 drugs approved by the FDA, 10 small molecule compounds with relatively high docking scores were screened out ( Figure 7 B).

[0055] Among them, hesperidin showed outstanding performance in the experiment of inhibiting the binding of YTHDF1 to OTUD5 mRNA in CAFs, showing a significant inhibitory effect ( Figure 7 C).

[0056] As a natural compound with various biological activities such as antioxidant, antiviral, and antitumor, hesperidin is an important target in drug research and development. To further confirm the interaction between hesperidin and YTHDF1, an immunofluorescence experiment was conducted, and the results showed that biotin-labeled hesperidin (Biotin-HP) was significantly co-localized with YTHDF1 in CAFs ( Figure 7 D). Fluorescence spectroscopy analysis also showed that hesperidin could interact with YTHDF1 in a dose-dependent manner ( Figure 7 E). Through surface plasmon resonance (SPR) experiment, the strong affinity between hesperidin and YTHDF1 was further verified (KD = 5.604E-6, Figure 7 F).

[0057] Molecular dynamics simulation analysis showed that the binding energy between hesperidin and YTHDF1 was -8.713 kcal / mol, and five stable hydrogen bonds were formed with the SER396, CYS412, ASN441, ASP507, and THR508 residues of YTHDF1 ( Figure 7 G). Therefore, hesperidin may play its pharmacological role by directly binding to and inhibiting the activity of YTHDF1.

[0058] Immune checkpoint blockade (ICB) has opened up a new situation for cancer treatment. Considering the inhibitory effect of Tregs on the antitumor immune response, it was further explored whether targeting YTHDF1 could enhance the efficacy of anti-PD-1 therapy for HCC.

[0059] By establishing a mouse orthotopic HCC model and treating it with hesperidin combined with anti-PD-1 ( Figure 7H). The results showed that hesperidin alone could effectively delay tumor growth and reduce Tregs infiltration. More importantly, the combination therapy of hesperidin and anti-PD-1 showed a significant synergistic effect, and the combination therapy exhibited the best anti-tumor effect in inhibiting HCC progression compared with any single treatment group ( Figure 7 I-7J). These results indicated that hesperidin significantly enhanced the efficacy of anti-PD-1 therapy by targeting YTHDF1.

[0060] Figure 7 Among them, (A) Schematic diagram of the high-throughput virtual screening process based on lead compound identification and pharmacological validation; (B) 3D structures of the top 10 candidate compounds with higher docking scores; (C) Screening of candidate compounds targeting the inhibition of YTHDF1 in CAFs by YTHDF1 RIP-qPCR experiment; (D) IF co-localization of hesperidin and YTHDF1 in CAFs, scale bar = 10 μm; (E) Fluorescence spectroscopy experiment showed that hesperidin inhibited the binding of bis-ANS to YTHDF1 in a dose-dependent manner; (F) SPR experiment detected the direct binding affinity of hesperidin and YTHDF1; (G) 3D and 2D structures of the molecular dynamics simulation of hesperidin and YTH domain; (H) Regular observation of tumor growth ability by small animal visible light in the orthotopic HCC model of C57BL / 6 mice; (I) Quantitative analysis of luciferase activity in mice of each group; (J) Flow cytometry experiment analyzed tumor-infiltrating Tregs in mice of each group. **p < 0.01, ***p < 0.001.

[0061] In summary, this study focused on the key role of YTHDF1 in tumor progression in the tumor microenvironment, and particularly explored the regulatory mechanism of YTHDF1 on immune cells, especially Tregs, in the HCC microenvironment. The study first revealed that YTHDF1 promoted the translation of OTUD5 mRNA by regulating m6A modification, and then regulated the stability of IDO1 through deubiquitination, driving changes in the tryptophan metabolic pathway. This process provided support for tumor immune escape. In addition, through a virtual screening strategy, the research team identified hesperidin as a potential inhibitor of YTHDF1 and further verified its role in CAFs. Hesperidin exerted anti-tumor effects by directly binding to YTHDF1 and inhibiting its interaction with OTUD5 mRNA, and when hesperidin was used in combination with anti-PD-1 antibody, it showed a significant synergistic effect and enhanced the anti-tumor immunotherapy effect. This study provided a new perspective, revealing the role of YTHDF1 in HCC progression and proposing a therapeutic strategy targeting YTHDF1, especially its potential in immune checkpoint inhibition therapy.

[0062] The present invention has been described by the above related embodiments. However, the above embodiments are only examples for implementing the present invention. It must be pointed out that the disclosed embodiments do not limit the scope of the present invention. On the contrary, modifications and refinements made without departing from the spirit and scope of the present invention fall within the scope of patent protection of the present invention.

Claims

1. Application of YTHDF1 in regulating tryptophan metabolism to promote the formation of immunosuppressive microenvironment in hepatocellular carcinoma.

2. Use of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma, characterized in that: The YTHDF1 regulates the HCC immune microenvironment.

3. Use of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma, characterized in that: The YTHDF1 enhances tryptophan catabolism.

4. Use of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma, characterized in that: The YTHDF1 promotes the translation of OTUD5 mRNA, and OTUD5 directly interacts with IDO1 and inhibits its ubiquitination and degradation.

5. Use according to claim 1 of YTHDF1 in regulating tryptophan metabolism to promote the formation of an immunosuppressive microenvironment in hepatocellular carcinoma, characterized in that: Hesperidin improves the efficacy of anti-PD-1 therapy by targeting YTHDF1.

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