Kit for detecting clear cell renal cell carcinoma

Abstract

The invention relates to a kit for detecting clear cell renal cell carcinoma. The kit comprises: a) at least one reagent capable of inhibiting aldolase A (ALDOA) K230 site lactic acid modification; b) at least one agent capable of modulating the activity of histone acetyltransferase CBP (CREB binding protein); and / or c) at least one agent capable of modulating the activity of the deacetylase SIRT7.

Description

Technical Field

[0001] The present invention relates to a kit for detecting clear cell renal cell carcinoma. Background Art

[0002] Lysine lactation (Kla) in histones, a key epigenetic modification pathway, has been increasingly studied in recent years in the field of gene transcription regulation. Studies have shown that Kla not only has a significant impact on gene transcription through epigenetic mechanisms (Li et al., 2023; Li et al., 2022; Zhang et al., 2019), but also plays a role in regulating the function of target proteins in non-histone proteins, and is closely associated with malignant behaviors such as cell proliferation, invasion, colony formation, drug resistance, and attenuation of spontaneous apoptosis (Li et al., 2024b). Notably, Kla activity is positively regulated by high concentrations of lactate. Lactate, as the end product of glycolysis, is precisely controlled by alanyl-tRNA synthase 1 / 2, which has unique lactate transferase and delactase activities (Li et al., 2024b). Previous studies have further revealed that the increase in Kla sites enhances glycolysis / gluconeogenesis in tumors, leading to elevated lactate levels, thereby forming a positive feedback loop between lactation and glycolysis. However, little is known about the specific mechanisms by which specific Kla sites can be regulated by specific enzymes (i.e., "writers" and "transfer hands") during disease pathogenesis.

[0003] Clear cell renal cell carcinoma (ccRCC), the most common malignant tumor of the kidney, continues to rise in incidence worldwide, posing a serious threat to human health. Although surgical resection is the preferred treatment for malignant renal tumors, most patients are diagnosed in the late stages, missing the optimal treatment window and relying on traditional chemoradiotherapy. However, patients with advanced ccRCC often respond poorly to these traditional treatments, and the risk of postoperative tumor recurrence and distant metastasis is extremely high, resulting in a high mortality rate for the disease (Bukavina et al., 2022; Sung et al., 2021; Capitanio et al., 2019; Cotta et al., 2023; Hsieh et al., 2017).

[0004] With the advancement of medical research, targeted therapy has become an important treatment for advanced ccRCC. However, drug resistance remains a serious problem, greatly limiting treatment efficacy and adversely affecting patient prognosis (Atkins and Tannir, 2018; Riaz et al., 2021). Currently, there is an urgent need to develop new drugs to overcome the limitations of existing treatments and improve the survival rate and quality of life of ccRCC patients. However, due to the lack of effective therapeutic targets and specific biomarkers to assess patient response to treatment and prognosis, the treatment of ccRCC still faces huge challenges. Summary of the Invention

[0005] To address the above-mentioned deficiencies of the prior art, the present application provides a kit for preventing and / or treating clear cell renal cell carcinoma by performing extensive proteomic, phosphoproteomic, and lactomic analyses of pre-treatment ccRCC tumor samples and their corresponding normal adjacent tissues (NATs) from 49 Chinese patients.

[0006] The present invention is achieved by adopting the following technical solutions.

[0007] In one aspect, the present invention provides a kit for preventing and / or treating clear cell renal cell carcinoma, the kit comprising:

[0008] a) at least one agent capable of inhibiting lactylation modification at K230 of aldolase A (ALDOA);

[0009] b) at least one agent capable of modulating the activity of histone acetyltransferase CBP (CREB binding protein); and / or

[0010] c) at least one agent capable of modulating the activity of the sirtuin SIRT7.

[0011] The agent capable of inhibiting the lactation modification of the ALDOAK230 site may be any agent capable of inhibiting, for example, targeted inhibition of, the lactation modification of the ALDOAK230 site.

[0012] Preferably, the agent capable of regulating the activity of histone acetyltransferase CBP (CREB binding protein) refers to an agent capable of downregulating the activity of histone acetyltransferase CBP (CREB binding protein), i.e., a histone acetyltransferase inhibitor. The agent capable of downregulating the activity of histone acetyltransferase CBP (CREB binding protein) can be, for example, a CBP inhibitor (ICG-001), catalog number: Cat# HY-14428, purchased from MCE USA.

[0013] Preferably, the agent capable of regulating the activity of the deacetylase SIRT7 refers to an agent capable of upregulating the activity of the deacetylase SIRT7, ie, a deacetylase SIRT7 activator.

[0014] The reagent capable of upregulating the activity of the deacetylase SIRT7 can be, for example, a SIRT7 activator (Resveratrol), Cat# HY-16561, purchased from MCE USA.

[0015] The reagent capable of inhibiting the lactylation modification of the ALDOAK230 site (for example, it can be CRISPR gene editing), the reagent capable of downregulating the activity of histone acetyltransferase CBP (CREB binding protein) (for example, it can be a small molecule compound), and the reagent capable of upregulating the activity of deacetylase SIRT7 (for example, it can be a small molecule compound) can be selected from: small molecule compounds, monoclonal antibodies, antisense oligonucleotides, shRNA, siRNA, and at least one of the CRISPR gene editing system.

[0016] In another aspect, the present invention provides a kit for identifying the malignancy of clear cell renal cell carcinoma, the kit comprising a reagent for detecting lactylation modification of the K230 site of aldolase A (ALDOA).

[0017] The reagent for detecting the lactylation modification level of aldolase A (ALDOA) ​​K230 site can be purchased from Jingjie Biological Co., Ltd., product number: Cat#CN0927.

[0018] In the present invention, the malignancy of clear cell renal cell carcinoma can be determined by detecting the level of lactylation modification at the aldolase A (ALDOA) ​​K230 site. If the level of lactylation modification at the aldolase A (ALDOA) ​​K230 site is higher, the malignancy is higher; if the level of lactylation modification at the aldolase A (ALDOA) ​​K230 site is lower, the malignancy is lower.

[0019] Preferably, the kit is provided with a lactic acidification level grading standard:

[0020] If the positive rate of immunohistochemical staining is less than 30%, it is considered to be of low malignancy;

[0021] If the positive rate of immunohistochemical staining is 30%-60%, it is considered to be of moderate malignancy;

[0022] If the positive rate of immunohistochemical staining is greater than 60%, it is considered to be highly malignant.

[0023] Compared with the existing technology, this application has at least the following excellent technical effects:

[0024] Currently, kidney cancer is mostly diagnosed using enhanced CT scans, and occasionally biopsies and HE staining. However, these techniques are time-consuming, invasive, and inaccurate. The kit of the present invention can quickly and accurately determine the malignancy of kidney cancer by detecting the lactylation modification level of ALDOA-K230. This method offers the advantages of accuracy, time efficiency, and the fact that it does not require large tissue samples.

[0025] Based on the research of the present invention, agents that inhibit lactylation modification of aldolase A (ALDOA) ​​K230 site, downregulate the activity of histone acetyltransferase CBP (CREB binding protein), and upregulate the activity of deacetylase SIRT7 can prevent or treat clear cell renal cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :Multi-omics analysis of clear cell renal cell carcinoma (ccRCC). Among them:

[0027] (A) Workflow of multi-omics analysis for ccRCC screening.

[0028] (B) Demographic and clinical data of all samples and their multi-omics analysis.

[0029] (C) Lactomyome profile of ccRCC samples.

[0030] (D) Icelogo shows representative motif sequences of all lactoylation sites.

[0031] (E) Venn diagram showing the overlap of lactoylation sites and differentially expressed proteins between ccRCC and non-tumor adjacent tissues (NATs).

[0032] (F) Representative images of immunohistochemical analysis showing Ki67 expression in ccRCC tumors and their paired normal tissues (N = 49). The proportions of Ki67 expression at different levels in these tissues. P < 0.0001 as determined by the Mann–Whitney test. Scale bar = 200 μm (100X), or scale bar = 50 μm (400X).

[0033] Figure 2 : Proteomic and phosphoproteomic alterations in clear cell renal cell carcinoma (ccRCC) tumors compared with non-tumor adjacent tissue (NAT).

[0034] (A) Principal component analysis (PCA) of proteomic data from 49 paired ccRCC tumor and NAT samples.

[0035] (B) Principal component analysis of phosphoproteomic data from 49 paired ccRCC tumor and NAT samples.

[0036] (C) Heat map showing the differential expression of CT antigen proteins and phosphorylation sites in this cohort.

[0037] (D) Comparison of phosphorylation sites and changes in their corresponding protein abundance.

[0038] (E) Volcano plot showing the enrichment analysis results of post-translational modification (PTM) features of differentially expressed and phosphorylated proteins between tumors and matched NATs.

[0039] (F) Boxplots showing the responses of erlotinib to VHL-mutant and wild-type ccRCC cells.

[0040] (G) CausulPath displays the transcription factor (TF)-target gene (TG) regulatory network in ccRCC tumors.

[0041] Figure 3 : Characterization of histone and non-histone lysine lactylation in clear cell renal cell carcinoma (ccRCC).

[0042] (A) Principal component analysis (PCA) of the distribution of different levels of lysinophilic bacteria in 49 paired ccRCC tumor and non-tumor adjacent tumor (NAT) samples.

[0043] (B) Heat map showing the abundance of histones and their lysine lactylation between ccRCC and NATs.

[0044] (C) Relationship between histone lysine lactoylation sites and acyltransferases, deacylases, and bromodomain-containing proteins.

[0045] (D) Heat map showing the correlation between histone lysine lactylation and cancer pathway signatures. Correlations with p < 0.05 are indicated by asterisks.

[0046] (E) Volcano plot showing differentially expressed lactoylation sites.

[0047] (F) Scatter plot showing the identified lactylation sites and the differentially expressed lactylation sites in tumor-NAT.

[0048] (G) Correlation between the number of lactoylation sites and the abundance of the corresponding proteins.

[0049] (H) Functional enrichment analysis of tumor-NAT differentially expressed lactylated sites and corresponding proteins after adjusting for protein expression levels.

[0050] Figure 4 :CBP is ALDOA-K230 lactoylation acyltransferase. Among them:

[0051] (A) Immunoprecipitation and immunoblotting analysis of ALDOA lysine lactoylation in clear cell renal cell carcinoma (ccRCC) and non-tumor adjacent tissues (NATs).

[0052] (B) Myc-tagged ALDOA was overexpressed and cells were treated with or without 10 mM L-lactic acid. Subsequently, cells were immunoprecipitated with anti-Myc antibody and detected with anti-L-lactic acid lysine antibody.

[0053] (C, D) Immunoprecipitation and immunoblot analysis of ALDOA lysine lactoylation in Myc-ALDOA-overexpressing HEK293 cells.

[0054] (E) Flag-tagged P300, CBP, p300 / CBP-associated factor (PCAF), or Myc-tagged GCN5 were overexpressed, and Myc-tagged ALDOA was co-transfected. ALDOA was immunoprecipitated using anti-Myc antibody and detected using anti-L-lactate lysine antibody.

[0055] (F) Immunoprecipitation and immunoblotting analysis of the interaction between Myc-ALDOA and Flag-CBP in HEK293T cells.

[0056] (G) Immunoprecipitation and immunoblotting analysis of the interaction between Myc-ALDOA and Flag-CBP in HEK293T cells overexpressing Myc-ALDOA.

[0057] (H) Myc-tagged ALDOA cells were overexpressed and treated with or without a CBP inhibitor. Subsequently, cells were immunoprecipitated with anti-Myc antibody and detected with anti-L-lactate lysine antibody.

[0058] (I) Myc-tagged ALDOA was overexpressed and cells were treated with or without CBP activators. Subsequently, cells were immunoprecipitated with anti-Myc antibody and detected with anti-L-lactate lysine antibody.

[0059] (J) HEK293T cells were transfected with ALDOA wild-type or K230R (non-lactating mutant) plasmids, with or without CBP overexpression, and Myc-tagged ALDOA was immunoprecipitated for lactoylation analysis.

[0060] (K) Mass spectrometry (MS)-MS spectrum of ALDOA-K230 in ccRCC tumors. b and y indicate the N-terminus and C region of the peptide, respectively.

[0061] (L) Alignment of the sequences surrounding K230 in ALDOA homologs from different species. The lactylated lysine residue in ALDOA is highlighted (bold and red).

[0062] (M) ALDOA WT or K230R (non-lactating mutant) plasmids were transfected into HEK293T cells, and the cells were subsequently immunoprecipitated with anti-Myc antibody and detected using anti-ALDOA-K230-lactate-lysine antibody.

[0063] (N) ccRCC tissue and NAT were immunoprecipitated using ALDOA-K230-lactoyllysine antibody and detected with ALDOA.

[0064] (O) Myc-tagged ALDOA was overexpressed and treated with or without a CBP inhibitor. Subsequently, cells were immunoprecipitated with an anti-Myc antibody and probed with an anti-ALDOA-K230-lactyl-lysine antibody.

[0065] (P) Myc-tagged ALDOA was overexpressed and treated with or without a CBP activator. Subsequently, cells were immunoprecipitated with anti-Myc antibody and probed with anti-ALDOA-K230-lactate-lysine antibody.

[0066] (Q) HEK293T cells were transfected with ALDOA wild-type or K230R (non-lactating mutant) plasmids, with or without CBP overexpression, and Myc-tagged ALDOA protein was immunoprecipitated and detected using an anti-ALDOA-K230-lactate-lysine antibody for lactation analysis.

[0067] (R) Representative images of immunohistochemical analysis of CBP expression in ccRCC tumors and paired NATs (N=49). Scale bar = 200 μm (100X), or scale bar = 50 μm (400X).

[0068] Figure 5 :SIRT7 is a deamidase that is involved in the lactoylation of ALDOA-K230.

[0069] (A) SIRT1-SIRT7 were overexpressed separately and Myc-tagged ALDOA was co-transfected. ALDOA was immunoprecipitated with anti-Myc antibody and detected with anti-L-lactate lysine antibody.

[0070] (B, C) Immunoprecipitation and immunoblotting analysis of the interaction between SIRT7 and Myc-ALDOA in HEK293T cells overexpressing Myc-ALDOA.

[0071] (D) Myc-tagged ALDOA was overexpressed alone and co-transfected with a FLAG-tagged vector or FLAG-tagged SIRT7. ALDOA was immunoprecipitated with an anti-Myc antibody and detected with an anti-L-lactyl-lysine antibody.

[0072] (E) Cells were overexpressed with Myc-tagged ALDOA and treated with or without a SIRT7 inhibitor. Subsequently, cells were immunoprecipitated with anti-Myc antibody and detected with anti-L-lactate lysine antibody.

[0073] (F) Myc-tagged ALDOA cells were overexpressed and treated with or without SIRT7 activators. Subsequently, cells were immunoprecipitated with anti-Myc antibody and detected with anti-L-lactate lysine antibody.

[0074] (G) ALDOA WT or K230R (non-lactating mutant) plasmids were introduced into HEK293T cells with or without SIRT7 overexpression. Myc-tagged ALDOA was immunoprecipitated for lactation analysis.

[0075] (H) Myc-tagged ALDOA was overexpressed and treated with or without a SIRT7 inhibitor. Subsequently, the cells were immunoprecipitated with an anti-Myc antibody and detected with an anti-ALDOA-K230-lactoyllysine antibody.

[0076] (I) Myc-tagged ALDOA was overexpressed and treated with or without SIRT7 activators. Subsequently, cells were immunoprecipitated with anti-Myc antibody and detected with anti-ALDOA-K230-lactyl-lysine antibody.

[0077] (J) HEK293T cells were transfected with ALDOA wild-type or K230R (non-lactating mutant) plasmids, with or without SIRT7 overexpression, and Myc-tagged ALDOA protein was immunoprecipitated and detected using an anti-ALDOA-K230-lactate-lysine antibody for lactation analysis.

[0078] (K) Representative images of immunohistochemical analysis of SIRT7 expression in ccRCC tumors and paired NATs (N = 49). Scale bar = 200 μm (100X), or scale bar = 50 μm (400X).

[0079] Figure 6 : ALDOA-K230R molecular docking and proteomic analysis revealed the role of ALDOA in tumor proliferation.

[0080] in:

[0081] (AD) Supernatant glucose concentration, extracellular lactate concentration, intracellular ATP level, and ALDOA enzyme activity were measured in normal control (NC) and shALDOA786-O cells induced to express the exogenous vector control WT-ALDOANTm (synonymous) or K230R-ALDOANTm (synonymous) (n = 3 biological replicates).

[0082] (E) Structural changes of ALDOA upon K230 acylation.

[0083] (F) Docking poses of ALDOA and ACTG1. The upper panel shows the interaction between non-lactated ALDOA and ACTG1. The lower panel shows the interaction between K230-lactated ALDOA and ACTG1.

[0084] (G) Workflow design for proteomic analysis of ALDOA and ALDOA-K230R overexpressing 786-O cells.

[0085] (H) Proteins of ALDOA-K230R and ALDOA-WT cells were identified.

[0086] (I) Expression levels of ALDOA and ALDOA-K230R were similar in ALDOA-overexpressing and ALDOA-K230R-overexpressing cells used for proteomic analysis (left).

[0087] (J) Principal component analysis (PCA) of the proteomes of ALDOA-K230R and ALDOA-WT cells (right).

[0088] (K) Gene set enrichment analysis showed that ALDOA-K230R cells had impaired proliferation ability.

[0089] (L) Volcano plot showing differentially expressed proteins between ALDOA-K230R and ALDOA-WT cells (right).

[0090] (M) CD44 expression between ALDOA-K230R and ALDOA-WT cells.

[0091] (N) Stemness scores between ALDOA-K230R and ALDOA-WT cells.

[0092] Quantitative data are presented as mean ± standard deviation. Differences between groups were analyzed by two-way analysis of variance and post hoc Bonferroni multiple comparison test (AD, p values ​​adjusted for six comparisons). NS: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0093] Figure 7 : Re-induction of Myc-ALDOA overexpression, but not its K230R mutant, restored the malignant behavior of Myc-ALDOA-silenced 786-O cells in vitro.

[0094] (A) Western blot analysis of ALDOA silencing in 786-O cells, and the expression of ALDOA was detected using three shRNA fragments (126866, 126867, and 126868) (n = 3 biological replicates).

[0095] (B) Western blot showed that ALDOA silencing reduced the expression levels of vimentin, fibronectin, and N-cadherin in 786-O cells (n = 3 biological replicates).

[0096] (C) The expression levels of EMT-related markers were detected in shALDOA786-O cells that were forced to express exogenous vector control WT-ALDOANTm or K230R-ALDOANTm (n = 3 biological replicates).

[0097] (DG) The proliferation, migration, and invasion abilities of shALDOA786-O cells upon forced expression of exogenous vector control WT-ALDOANTm or K230R-ALDOANTm were analyzed using CCK8, scratch healing, Transwell invasion, and colony formation assays (n = 3 biological replicates).

[0098] (H) After knockdown of ALDOA, the expression levels of apoptosis-related markers were detected by western blot analysis (n = 3 biological replicates).

[0099] (I) The expression levels of apoptosis-related markers were detected in shALDOA 786-O cells that were forced to express exogenous vector control WT-ALDOANTm or K230R-ALDOANTm (n=3 biological replicates).

[0100] (J) Flow cytometric analysis of apoptotic cell rates in Myc-ALDOA-silenced 786-O cells (n=3 biological replicates).

[0101] Quantitative data are expressed as mean ± standard deviation. Relative protein levels were calculated as fold change relative to the reference group. Differences between groups were analyzed using unpaired t-tests (B, H) and two-way ANOVA with post hoc Bonferroni multiple comparison tests (C, I, p values ​​adjusted for three comparisons; A, E–G, J, p values ​​adjusted for six comparisons). IB: immunoblot; NS: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0102] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0103] Example

[0104] 1. Methods

[0105] 1. Tissue Sample Preparation

[0106] Individual human ccRCC or NAT tissue samples were prepared for proteomic analysis. Specifically, tissue samples were ground into a powdered homogenate in liquid nitrogen and suspended in four volumes of lysis buffer (1% Triton X-100, 1% protease inhibitor cocktail, 1% phosphatase inhibitor, 3 μM tailorase A (TSA), 50 mM nicotinamide (NAM)), followed by brief sonication on ice. After centrifugation, the supernatant was collected and protein concentration was determined using a BCA kit (Shanghai Biotech Co., Ltd.) according to the manufacturer's instructions.

[0107] Equal amounts of protein samples were digested with trypsin at a 1:50 ratio (protease:protein) at 4°C overnight. Proteins were slowly precipitated by the addition of 20% trichloroacetic acid, mixed, and kept at 4°C for 2 hours, followed by centrifugation to obtain a protein plate. After washing 2-3 times with pre-cooled acetone, the protein plate was air-dried and solubilized in 200 mM TEAB by gentle sonication. Subsequently, the plate was reduced with 5 mM dithiothreitol (DTT) at 56°C for 30 minutes at room temperature in the dark, followed by alkylation with 11 mM iodoacetamide (IAA) for 15 minutes. Finally, the peptides were analyzed by conventional HPLC-MS on an I-Class TUVWFC system (Waters Corporation, MA, USA).

[0108] 2. Database search

[0109] MS / MS data were processed using the MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the human Swiss-Prot database (20,376 records, 2022.01.07) and linked to a reverse bait database. Trypsin / P was designated as the cleavage enzyme, allowing up to two missed cleavage sites for proteome and phosphoproteomic analyses, while four missed cleavage sites were allowed for lactoylomic analyses. The mass tolerance for both precursor and fragment ions was set to 20 ppm for both the first and main searches. Carboxymethylation on cysteine ​​was defined as a fixed modification, while acetylation at the protein N-terminus and oxidation on methionine were designated as variable modifications. For phosphoproteomic samples, phosphorylation on serine / threonine / tyrosine was used as an additional variable modification. For lactoylomic samples, lactoylation on lysine was used as an additional variable modification. Proteins with at least one unique peptide and an FDR below 1% at both the peptide and protein levels were selected for further analysis.

[0110] 3. Preprocessing of proteomics, phosphoproteomics, and lactoylation data

[0111] Proteomic quantification was performed using the iBAQ algorithm, and the phosphoproteomics and lactoylation groups were quantified at the site level. All quantitative data were normalized to the median of each sample to correct for potential sample loading differences and multiplied by the global median of each omics dataset for ease of presentation and log-2 transformed. Proteins, phosphorylation sites, and lactoylation groups with >50% missing values ​​in tumor and normal control samples were excluded from further analysis. K-nearest neighbor (KNN) imputation was performed using the R package DreamAI to fill missing values. (Ma et al., 2020) (doi: https: / / doi.org / 10.1101 / 2020.07.21.214205) Tumor and NAT samples were analyzed separately.

[0112] 4. Protein and pathway changes in tumors and adjacent tissues

[0113] Volcano plots were used to display differentially expressed proteins and their modifications in tumor and NAT samples by applying a threshold of FC>2 and BH correction p<0.05. Proteins and their modifications that were significantly upregulated or downregulated in tumor tissues were subjected to functional enrichment analysis using ConsensusPathDB ( http: / / cpdb.molgen.mpg.de / )(Kamburov et al., 2011).(21071422)

[0114] 5. Kinase activity detection

[0115] Kinase activity was inferred based on predicted substrates from kinase-substrate relationships obtained from PhosphoSitePlus and NetworKIN (Linding et al., 2007) (17570479). Substrates with a NetworKIN score > 5 were retained. The kinase activity of each sample was calculated using the single-sample gene set enrichment analysis (ssGSEA) method in the GSVA package. Kinase-substrate enrichment analysis (KSEA) was performed using the PhosphoSitePlus and NetworKIN databases and their KSEA application (https: / / casecpb.shinyapps.io / ksea / ). (Wiredja et al., 2017) (28655153) Statistical significance was set at an FDR < 0.05.

[0116] 6. Transcription factor (TF) activity detection

[0117] TF targets were retrieved from DoRothEA (Garcia-Alonso et al., 2019) (31340985), and only targets with "A" or "B" confidence levels were used for analysis. The pre-processed proteomic data were analyzed using the VIPER package to infer the transcription factor activity of each sample (Alvarez et al., 2016) (27322546). Differences in inferred activity between tumor and non-tumor control samples were statistically analyzed using the Wilcoxon rank sum test, with BH-corrected p values ​​< 0.05 considered significant.

[0118] 7. PTM-SEA

[0119] PTM-SEA (Krug et al., 2019) (30563849) was performed to identify dysregulated phosphorylation-driven pathways. The PTM signature database (PTMsigDB) v1.9.1 was downloaded from https: / / github.com / broadinstitute / ssGSEA2.0 / tree / master / db / ptmsigdb / v1.9.1 / . PTM-SEA was performed on the GenePattern platform (https: / / cloud.genepattern.org / ) (Reich et al., 2006) (16642009) using Defauts.

[0120] 8. Analysis of Erlotinib Response

[0121] VHL mutation status and area under the drug response curve (AUC) for erlotinib in ccRCC cells were obtained from DepMap (Prism Repurposing Secondary Screening). https: / / depmap.org / portal / ).

[0122] 9. Relationship between lactoylation and acyltransferase and deacylase

[0123] Spearman correlation tests were performed to compare the levels of lactoylation sites with acyltransferase and deacylase levels. Enzyme-lacoylation site pairs with BH-corrected p-values ​​< 0.05 were considered as potential regulatory relationships.

[0124] The abundance values ​​of histone lactylation from different histone gene homologs of a functional site were averaged. To test the association between acyltransferase / deacylase and histone lactylation levels, a lasso regression model was fitted using acyltransferase / deacylase and histone protein expression as independent variables and histone lactylation sites as dependent variables. (Dou et al., 2020)(32059776). 300 bootstraps were performed using 80% of the training data and 20% of the test data, and the average coefficient returned by the model over 300 iterations is reported. Proteins with an average coefficient < 0.1 were discarded.

[0125] 10. Cell culture

[0126] Human ccRCC 786-O cells were purchased from the Cell Bank of the Chinese Academy of Sciences' Center for Microbial Culture Collection and authenticated by STR analysis. Cells were cultured in Roche-Parkinson Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS). HEK293T cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, penicillin (100 U), and streptomycin (100 μg / ml). Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0127] 11. Plasmid construction and transfection

[0128] Full-length human ALDOA and ALDOA DNA fragments carrying WT-NTm (silent nucleotide mutation) or K230R-NTm mutants, which prevent potential in vivo clearance, as well as different lysine mutation sites (K13R, K42R, K99R, K108R, K230R, K312R, and K330R) (GeneChem, Shanghai, China) were cloned into the 1X Myc GV712 vector. 1X Flag-SIRT1-7 plasmids, Flag-P300, Flag-CBP, and Myc-GCN5 plasmids, and pCI-FLAG-PCAF plasmid were purchased from GeneChem. Plasmid transfection was performed using Lipofectamine 3000 (Cat. No. L3000015, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Cells were harvested 36 to 48 hours after transfection.

[0129] 12. Lentivirus Preparation and Transduction

[0130] Lentivirus expressing ALDOA-specific shRNA was purchased from GeneChem to establish an ALDOA-silencing stable cell line (Gardiner et al., 2020). Briefly, lentiviral particles were collected from the supernatant of cultured HEK293T cells according to the manufacturer's instructions (Gardiner et al., 2020). 786-O cells were cultured overnight in 6-well plates and then transduced with control or ALDOA-shRNA-expressing lentivirus at an MOI of 10 in 5× PEG-it™ solution (Cat. No. LV810A-1, SystemBiosciences, Johnstown, PA, USA). Cells were treated with puromycin (10 μg / ml) for seven days to establish a stable cell line. Finally, the effect of ALDOA silencing in cells was examined by Western blot analysis.

[0131] 13. Preparation of anti-ALDOA-K230 lactate lysine rabbit polyclonal antibody.

[0132] Specific polyclonal antibodies were routinely generated by PTM Bio (Hangzhou, China). Briefly, two ALDOA-K230 lactoylated peptides and a control WT-ALDOA-K230 peptide were designed and synthesized based on their protein sequence. The ALDOA-K230 lactoylated peptides were coupled to the keyhole limpet hemocyanin (KLH) carrier protein. Six New Zealand rabbits were immunized multiple times with the ALDOA-K230 lactoylated peptide / KLH in 50% FA or IFA, and their sera were then tested by ELISA and Western blot against the immunized ALDOA-K230 lactoylated peptide or the unlactated ALDOA control peptide. Subsequently, anti-ALDOA-K230 lacto-lysine rabbit serum purified from the immunogenic peptide was pre-adsorbed. The purified polyclonal antibodies were characterized by Western blot for their specific recognition of ALDOA-K230la but not WT-ALDOA.

[0133] 14. Lactation test

[0134] Cells were harvested after 24 hours of treatment with or without L-lactic acid (10 mM, Sigma, USA) and lysed in lysis buffer for immunoprecipitation and Western blot analysis. Additionally, some cells were treated with the SIRT7 inhibitor 97491 (MedChemExpress, Monmouth County, MD, USA) at 5 μM for 24 hours or with the SIRT7 activator resveratrol (MedChemExpress, Monmouth County, MD, USA) at 50 μM for 10 hours. Furthermore, some cells were treated with the CBP-specific activator TTK21 (MedChemExpress, Monmouth County, MD, USA) at 50 μM or ICG-001 (MedChemExpress, Monmouth County, MD, USA) at 50 μM for 24 hours. ALDOA lactylation levels in cells from different groups were analyzed.

[0135] 15. Western blotting and immunoprecipitation

[0136] Relative levels of target protein expression were quantified by Western blotting as previously described (Terui et al., 2003) (14695212). Data were analyzed using ImageJ software. For immunoprecipitation, different cell groups were collected and washed three times with phosphate-buffered saline (PBS) and then lysed using flag lysis buffer (137 mM sodium chloride, 10 mM sodium fluoride, 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 0.1 mM Na3S, 10% glycerol, 1% nonionic detergent P-40 (NP-40), and 1 mM PMSF). Cell lysates were incubated with anti-c-Myc magnetic beads at 4°C for 12 hours or with the corresponding antibodies at 4°C for 3 hours, followed by reaction with protein A / G magnetic beads at 4°C for 12 hours. After washing, the protein-antibody complexes were eluted with 4x SDS loading buffer.

[0137] 16. Human specimens

[0138] This study was approved by the Research Ethics Committee of Shengjing Hospital, China Medical University (No. AF-S0P-07-1.1-01). Forty-nine human surgically resected ccRCC tissues and adjacent non-tumor tissues were obtained from the Department of Urology, Shengjing Hospital, China Medical University (Shenyang, China). Individual patients were diagnosed with ccRCC after histological confirmation according to the guidelines of the Declaration of Helsinki. All patients provided written informed consent before surgery.

[0139] 17. Immunohistochemistry of human kidney tissue sections

[0140] The expression level of target proteins was characterized by immunohistochemistry, as described in the report (Ji et al., 2016; Lin et al., 2022). Briefly, surgical kidney tissue samples were fixed with formalin and paraffin-embedded. After deparaffinization and rehydration, kidney tissue sections (4 pm) were subjected to antigen retrieval. After blocking, the sections were incubated with primary antibodies against CBP (Cat# 7389S, Cell Signaling Technology, Boston, MA, USA), SIRT7 (Cat# 12994-1-AP, Proteintech, Wuxi, China), ALDOA (Cat# PTM-5790, PTMab, Hangzhou, China), ALDOA-K230 site-specific antibody (Cat# CN0927, PTMab), and Ki67 (Cat# 27309-1-AP, Proteintech) at 4°C overnight. After washing, the sections were reacted with biotin-labeled IgG in a hypersensitive SPIHC kit (Cat# KIT-9710, MXB, Qingdao, China) and developed with DAB (Cat# DAB-0031, MXB), followed by hematoxylin counterstaining. The signal intensity scoring criteria were as follows: 0: no positive staining (negative); 1: light yellow (weakly positive); 2: brownish yellow (positive); 3: brown (strongly positive). In addition, the percentage of positive cells scoring criteria were as follows: 1: < 25%; 2: 26%-50%; 3: 51%-75%; 4: > 75%. The final image score was calculated by multiplying the staining intensity score with the percentage of positive cells.

[0141] 18. Cell proliferation assay

[0142] Cell proliferation was determined by CCK-8 assay. Briefly, different groups of cells (3 x 10 3 were cultured in 96-well plates for different time periods in triplicate. 10 pl of CCK8 solution was added to each well, and incubation was continued for 2 hours. The absorbance of each well at 450 nm was measured using an absorbance reader (TECAN, Switzerland).

[0143] 19. Transwell assay

[0144] The effect of ALDOA expression change or ALDOA K230 mutation on ccRCC cell invasion was determined by transwell invasion assay. Different groups of ccRCC 786-O cells (5 x 10 3Cells (cells / well) were placed in FBS-free RPMI1640 medium and loaded into the top chamber of a 24-well transwell plate (8-μm pores, Corning, USA) pre-coated with Matrigel. The bottom chamber was filled with RPMI1640 medium supplemented with 10% FBS. After 24 hours of culture, cells on the upper membrane of the top chamber were removed, and the invasive cells on the lower surface of the membrane of the top chamber were fixed with 4% paraformaldehyde solution. Subsequently, the plates were stained with crystal violet (Solarion, China) and photographed. Invasive cells were quantified using ImageJ software (ImageJ 1.45).

[0145] 20. Cell apoptosis detection

[0146] The percentage of apoptotic cells was analyzed by flow cytometry. Briefly, different cell groups (2 × 10 5 Cells / tube) were stained with 2 μl Annexin-V-FITC (20 μg / ml) in 100 μl binding buffer on ice for 30 min, followed by 1 μl propidium iodide (PI) (50 μg / ml). Cells were analyzed by flow cytometry using FlowJo software.

[0147] 21. Wound healing test

[0148] Different ccRCC 786-O cell populations were cultured in 6-well plates. When cells reached confluence, the cell monolayer was wounded using a micropipette tip. After the cells were cultured for an additional 24 hours, the wells were photographed. The wound healing area was calculated using ImageJ software.

[0149] 22. Colony formation test

[0150] Different cell populations (1×10 5 The cells were cultured in 6-well plates coated with 1.2% agarose for 10-14 days. The number of cell clones was counted under an inverted microscope, and the colony formation rate was calculated.

[0151] 23. Lactate and glucose concentration assessment

[0152] ccRCC 786-O cells (3.2×10 6Cells (100 cells / well) were cultured in 6-cm cell culture dishes and transfected with a control plasmid, ALDOA-WT-NTm (silent nucleotide mutation), or K230R-NTm plasmid using lipofectamine 3000. After 24 hours of culture, the supernatant was collected and the concentrations of lactate and glucose were determined using a lactate assay kit (product number KTB1100, Abbkine, Wuhan, China) and a glucose assay kit (product number KTB1300, Abbkine, Wuhan, China) according to the manufacturer's instructions. Lactate and glucose concentrations were measured at 450 nm and 630 nm, respectively, using an absorbance reader (TECAN, Switzerland).

[0153] 24. ATP concentration and ALDOA enzyme activity determination

[0154] ccRCC 786-O cells were cultured in 6-cm dishes and transfected or not with the sh+ vector, ALDOA-WT-NTm, or ALDOA-K230R-NTm plasmids. After 24 hours of culture, supernatants were collected, and ATP concentration and ALDOA enzyme activity were measured in these supernatant samples using an ATP content assay kit (Cat. No. KTB1019, Abbkin, Wuhan, China) and an aldolase activity assay kit (Cat. No. ab196994, Abcam, Cambridge, UK), following the manufacturer's instructions. ATP concentration was measured at 560 nm using a BioTek Cytation 5 cell image multi-reader. ALDOA enzyme activity was measured at 450 nm.

[0155] 25. Relative value

[0156] Western blot images were analyzed using ImageJ software. The intensity of the internal reference band was used as a reference for measuring the intensity of the protein-loaded sample band. First, in an independent experiment, the relative expression of the target protein was calculated by dividing the grayscale value of each lane by the grayscale value of the corresponding internal reference, and then dividing the relative expression of each lane by its average. This calculation process was repeated independently. Secondly, the proportions of the first group (control group) calculated in the previous step were averaged, and then each proportion of the first group was divided by this average to obtain a relative value. Finally, the proportions of the other groups were also divided by this average to obtain the final value used for statistical analysis.

[0157] 26. Statistical Analysis

[0158] Data are expressed as mean ± standard deviation (SD). Data distribution was analyzed using the Shapiro-Wilk test, and homogeneity of variance was analyzed using the F test (for comparisons of two groups) or the Bartlett test (for comparisons of more than two groups), with a threshold p value of 0.05. Differences between groups with normal distribution and equal variance were analyzed using a two-tailed unpaired t-test, and differences between multiple groups were analyzed using a two-way analysis of variance followed by the Bonferroni multiple comparison test. Differences between groups with skewed data were analyzed using the Mann-Whitney test, and differences within groups with skewed data were analyzed using the Kruskal-Wallis test followed by the Dunn multiple comparison test. Bonferroni correction was applied to multiple comparisons to adjust for type I error inflation, and p-values ​​were corrected for multiple comparisons when necessary. The significance assessment method and the number of biological replicates for each experimental group are detailed in the figure legend. All statistical analyses were performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism software (version 9.0; GraphPad, Bethesda, MD, USA). P values ​​< 0.05 were considered statistically significant. Representative images reflect the mean results of each experiment and are used in relevant figures. 'n' in the figure legends indicates the number of biological replicates.

[0159] 2. Results

[0160] 1. Multi-level proteomic analysis of renal tissues in ccRCC patients

[0161] This study collected data from 49 Chinese patients with newly diagnosed ccRCC tumors and their paired non-tumor adjacent renal tissues (NATs). Their demographic and clinical characteristics are shown in Figure 1 B. As expected, immunohistochemistry showed that the positive rate of Ki67 expression in ccRCC tissues was significantly higher than that in NATs ( Figure 1 F). These 49 paired tissue samples and HeLa cell quality control (QC) samples were subjected to proteomics, phosphoproteomics, and lactoylation analyses using a mass spectrometry (MS)-based label-free quantitative strategy ( Figure 1 A, B). First, the analysis results of the QC samples yielded a Spearman median correlation coefficient of 0.966 (Figure S1A), demonstrating the consistency and stability of the mass spectrometry platform. Proteomic analysis identified 4,889 to 6,568 proteins in each sample, for a total of 7,735 proteins (Figure S1B). Phosphoproteomics and lactoylationomics analyses revealed an average of 9,223 phosphorylation sites and 2,490 Kla sites per sample, for a total of 34,359 unique phosphorylation sites and 10,968 unique Kla sites across all samples ( Figure 1C, Figure S1B). Of these, 1661 proteins were identified by all three methods. Microplate method (Figure S1C). The abundance of proteins in individual samples was quantified using the iBAQ algorithm and normalized to the median of each sample (Figure S1D). Similarly, the number of phosphorylation sites and lactylation sites was quantified and normalized to the median of each sample to correct for potential differences in sample loading (Figure S1D). To investigate the lactylation motif, potential amino acid lactylation sites were characterized and compared with the human proteome, revealing the presence of significant alanine and glycine around the modification sites, while cysteine ​​and serine were less abundant ( Figure 1 D), which is similar to the results of previous studies on the lactylation group. (Duan et al., 2024; Yang et al., 2023) (36593272, 39018247). In addition, a high proportion of Kla sites and Kla proteins were specifically detected in tumor samples and NATs, and the number of Kla sites and Kla proteins in tumors was greater than that in NATs ( Figure 1 E). Among tumor-specific Kla sites, 38 were identified in more than half of the tumor samples and enriched in extracellular exosomes (Figure S1E). Furthermore, 111 Kla sites were specifically identified in >50% of NAT samples and enriched in oxidative phosphorylation-related and kidney-specific proteins (Figure S1E). Clearly, differences in proteomic, phosphoproteomic, and lysylation profiles were observed between ccRCC tissue and NATs in this population.

[0162] 2. Proteomic and phosphoproteomic characteristics of ccRCC

[0163] To comprehensively understand the proteomic and phosphorylation characteristics of ccRCC, we further analyzed 6,653 proteins and 10,534 phosphorylation sites that were present in >50% of tumor or NAT samples. Principal component analysis (PCA) showed that the distributions of these proteins were significantly different between tumors and NATs at the proteomic and phosphorylation levels ( Figure 2A, B). A high degree of heterogeneity in tumor samples was also detected, which is consistent with previous reports. (Hu et al., 2024a; Qu et al., 2022) (35440542, 38361033) Compared with NATs, there were 2,576 differentially expressed proteins (DEPs, defined as a fold change (FC) > 2 and a Benjamin-Hochberg (BH)-corrected p-value < 0.05), including 1,043 downregulated and 899 upregulated proteins. In addition, 3,413 phosphorylation sites showed differential phosphorylation (BH-corrected p-value < 0.05, fold change FC > 2), 601 of which were less phosphorylated and 2,812 were more phosphorylated (Figure S2A, B). Interestingly, the upregulated DEPs included cancer testis-ovary (CT) antigen proteins (KIAA0100, POTEE, and SPAG9), some of which had unique phosphorylation sites in ccRCC tumors (GPATCH2 S115; KIAA0100 S2094, T1269; SPAG9 S183, T217, and S493) ( Figure 2 C). Among them, SPAG9 S183 and T217 phosphorylation were significantly enhanced, accompanied by increased expression and phosphorylation of α-synuclein-telangiectasia mutated (ATM) kinase in tumors. Further analysis showed that these DEPs were enriched in ccRCC tissues to enhance HIF signaling, glycolysis, DNA replication, and immune-related pathways, but weaken the tricarboxylic acid (TCA) cycle, amino acid metabolism, and fatty acid metabolism (Figure S2D). A large number of studies have shown that differentially phosphorylated phosphorylation sites tend to enhance glycolysis, proliferation, and immune response, but inhibit glucose oxidative phosphorylation, amino acid and fatty acid metabolism ( Figure 2 D). Multiple sites with known functional annotations (e.g., EGFR at S1166, PAK1 at T212, and MAPK1 at Y187) were highly phosphorylated in ccRCC tumors. PTM signature enrichment analysis (PTM-SEA) and kinase substrate enrichment analysis (KSEA) revealed the associated perturbations, kinase activities, and signaling pathways of these differentially expressed and differentially phosphorylated proteins. Figure 2 E, Figure S2E), especially the high activation of MAPK signaling pathway and cell cycle-related kinases. Several tyrosine kinase inhibitor (TKI) signature proteins were inactivated in tumors ( Figure 2 E). The perturbation signatures of EGF and EGFR inhibitors were upregulated and downregulated in tumors, respectively. In addition, in vitro experiments showed that VHL wild-type ccRCC cells were more sensitive to erlotinib than VHL mutant ccRCC cells ( Figure 2F) Next, transcription factor (TF) activities and their potential targets in tumors and NATs were analyzed using VIPER. Activated HIF1A increased. STAT1, STAT2, E2F4, and TP53 expression was upregulated, while HNF4A and VDR activation was decreased, which were predicted in tumors. Based on proteome, phospho-proteome, kinase activity, and transcription factor activity, a complete regulatory network was constructed using CausalPath (Figure S2G). By the maximum clique centrality (MCC) algorithm, the 10 hub nodes with the highest MCC values in the network were determined. Subsequently, two core oncogenic subnetworks were constructed, centered on NFKB1-RELA-SP1-TP53 and MAPK1-MAPK3-MAPK14-JUN common signaling events, respectively Figure 2 G) Therefore, ccRCC tumors have unique proteome and phospho-proteome features compared to native transcription factors.

[0164] 3. Changes in lactylation in ccRCC

[0165] Like other solid malignancies, ccRCC is more inclined to undergo glucose metabolism through the Warburg effect and contains high levels of lactate (Hakimi et al., 2016) (26766592). Principal component analysis of protein lactylation revealed a significant difference in the number of lysine (K) lactylation sites between ccRCC tumors and NATs Figure 3 A) Histone lactylation is an epigenetic regulator that can promote ccRCC progression through the PDGFRbeta signaling pathway (Yang et al., 2022) (35637958). Compared with NATs, Kla sites in histones and non-histones were significantly increased Figure 3 B), Kla sites in different histones were changed in ccRCC tumors (Figure S3A). Interestingly, the number of H2B K6 lactylation in elderly patients (age > 60 years) was significantly higher than that in young patients (Figure S3B). The expression of several acyltransferases and deacylases, which carry differentially lactylated K residues, showed ubiquitous enzymatic K lactylation in ccRCC (Figure S3C). In addition, the number of histone lactylation sites was positively correlated with the levels of acyltransferase, bromodomain-containing protein (BRD), and deacylase proteins Figure 3C, Figure S3D). The number of several histone Kla sites also positively correlated with the expression level of HDAC2 in ccRCC tumors, suggesting a compensatory increase in HDAC2 expression to regulate the "lactation-delactation" balance. (Yang et al., 2023) (36593272) In addition, the level of histone lactylation was negatively correlated with the expression level of SIRT5 in ccRCC tumors. In addition, core histone lactylation sites were positively or negatively correlated with the scores of signature pathways with potential for epigenetic regulation of histone lactylation ( Figure 3 D) Similarly, histone Kla sites are positively correlated with the regulation of proliferation-related pathways, suggesting that histone lactylation may promote ccRCC growth.

[0166] Compared with NATs, 490 Kla sites were highly lactylated and 590 sites were less lactylated in tumors (Benjamini-Hochberg corrected p value < 0.05, FC > 2) ( Figure 3 E), including 25 highly lactylated sites in histones. Thus, non-histone proteins carry the majority of altered lactylated Kla sites in tumors, with individual proteins ranging from 1 to 27 Kla sites. Eleven highly lactylated proteins were identified, with more than 10 Kla sites in ccRCC tumors, including ENO1, PGK1, ALDOA, and PKM involved in glycolysis ( Figure 3 F). Hyperlactylated and hypolactylated Kla sites in tumors exhibited distinct motifs (Figure S3E). Hyperlactylated Kla sites contained glycines at the +1 and -1 positions, whereas hypolactylated Kla sites contained alanines at multiple positions (Figure S3E). Pathway analysis revealed that hypolactylated Kla sites in tumors were primarily enriched in mitochondrial and metabolic pathways. In contrast, hyperlactylated Kla sites in tumors were primarily enriched in HIF-1 signaling and antigen processing and presentation pathways ( Figure 3 C). Interestingly, both high and low lactoylated Kla sites are enriched in glycolysis / gluconeogenesis ( Figure 3 C), highlighting the importance of protein K-lactylation in tumor glycolysis. Given that the quantification of protein lactylation levels is affected by protein abundance, the lactylation levels were normalized to the corresponding protein expression levels in tumors ( Figure 3 G). The number of K-lactylation sites in proteins is positively correlated with the corresponding overall protein expression level in tumors. Similarly, regardless of protein expression level, high- and low-lactylation K-lactylation sites are enriched in similar pathways in tumors ( Figure 3H). The number of K-lactylation sites in tumor proteins is also influenced by enzymes such as acyltransferases and deacylases. The number of K-lactylation sites correlated with the expression levels of certain enzymes (Figure S3E). Notably, EP300, ATF2, KAT7, NAA10, and NAA15 appear to be the most effective acyltransferases, while SIRT3, SIRT5, and HDAC6 are the most efficient deacylases in ccRCC. Thus, histone lactylation is crucial for tumor growth, while non-histone K-lactylation appears to regulate glycolysis and other metabolic pathways in ccRCC.

[0167] 4. CBP is ALDOA-K230 lactoacyltransferase

[0168] ALDOA is one of the important enzymes in the glycolysis process (Chang et al., 2018)(29907340). The lactoylation data of the present invention revealed that ALDOA has more than 1,000 lactoylation sites in ccRCC ( Figure 3 F). Bioinformatics analysis showed that ALDOA has K13, K42, K99, K108, K230, K312, and K330 sites within the glycolytic region (amino acids (aa) 14-363) (Figure S4A), which are critical for its enzymatic activity. Therefore, it was hypothesized that these sites may be lactylated in ccRCC. To verify this, the K site lactylation of ALDOA protein was searched in vitro. Consistently, immunoprecipitation revealed that the level of lactylated ALDOA protein in ccRCC tissues was significantly higher than that in NATs tissues ( Figure 4 A). After induction of Myc-tagged wild-type (WT) ALODA overexpression, anti-Myc immunoprecipitation showed that Myc-ALDOA was overexpressed, and L-lactic acid treatment slightly increased the level of lactoylated Myc-ALDOA in 786-O cells ( Figure 4 B). After inducing overexpression of Myc-tagged WTALDOA in HEK293T cells, anti-pan K-site precipitated increased levels of lactoylated Myc-ALDOA, and anti-Myc also precipitated higher levels of K-site lactoylated Myc-ALDOA ( Figure 4C, D). Given the role of ALDOA lactoylation in the progression of ccRCC, the enzymatic mechanism of ALDOA lactoylation was determined. To identify which acyltransferase(s) act on ALDOA, HEK293T cells were co-transfected with a plasmid expressing Flag-acyltransferase p300 (E1A binding protein, 300 kDa), Flag-CBP (CREB binding protein), Flag-PCAF (lysine acyltransferase 2B; p300 / CBP-associated factor), or Flag-GCN5 (histone acyltransferase GCN5). The results showed that inducing Flag-CBP overexpression significantly increased the K-site lactoylation level of ALDOA in this experimental system ( Figure 4 E). Immunoprecipitation showed that anti-Flag antibody precipitated Myc-ALDOA protein, highlighting the physical interaction between CBP and ALDOA and suggesting that Flag-CBP may be the factor responsible for the lactoylation of the ALDOAK site ( Figure 4 F). Histological examination showed that CBP expression level was high in ccRCC tissues and the ratio was higher than that of NATs ( Figure 4 R). Since SIRT7 has been identified as the delactylase of ALDOA ( Figure 5 ). In order to identify the K site of ALDOA lactoylation, K13, K42, K99, K108, K230, K312 or K330 were mutated to arginine (R, a non-lactoylation mutation) in Myc-WT-ALDOA by site-directed mutagenesis. To test their function, HEK293T cells were co-transfected with plasmids expressing Myc-ALDOA or ALDOA-KR mutants, and Flag control plasmid or Flag-SIRT7 plasmid was added. The results showed that transfection of ALDOA-K230R significantly reduced the lactoylation level of the Myc-ALDOAK site compared with cells transfected with Myc-WT-ALDOA. Regardless of the presence or absence of SIRT7, ALDOA-K230 remained at a very low level and no longer decreased (Figure S4B). ALDOAK230 is an evolutionarily conserved site from chickens to mammals ( Figure 4 L), which may be the main acetylation site in ALDOA and is regulated by CBP. Subsequently, an antibody specific for ALDOA-K230 lactoylysine was prepared and the level of ALDOA-K230 lactoylation was detected using this antibody, which was similar to the results using the pan-lactoyl-lysine antibody ( Figure 4 M). In addition, the ALDOA-K230 lactoylation level in ccRCC tissues was higher than that in NATs ( Figure 4Furthermore, in HEK293T cells, only Flag-CBP overexpression increased the levels of Myc-ALDOA and ALDOA-K230 lactoylation, but not Myc-ALDOA-K230R lactoylation, regardless of whether a pan-lactylation antibody or an ALDOA-K230 lactoylysine-specific antibody was used ( Figure 4 Finally, treatment with the CBP activator TTK21 enhanced the lactoylation of Myc-ALDOA and Myc-ALDOA-K230, whereas treatment with the CBP inhibitor reduced the lactoylation of Myc-ALDOA and Myc-ALDOA-K230 in vitro ( Figure 4 HI, OP). Therefore, CBP may be the acyltransferase for ALDOAK230 lactoylation.

[0169] 5. SIRT7 is a delactylase for ALDOA-K230

[0170] To further explore the regulatory mechanism of ALDOA-K230 delactylase, we identified the relevant ALDOA-K230 delactylase. The mammalian deacetylase family is classified as class III histone deacetylases (HDAC III), which consists of seven members (SIRT1-7), most of whose substrates are non-core histones (Vassilopoulos et al., 2011)(21807603). Therefore, we tested whether SIRT1-7 could delactylate ALDOA K230.

[0171] The results showed that induction of Flag-SIRT7 overexpression, but not its delactylase, significantly reduced the level of K-lactylating Myc-ALDOA in HEK293T cells ( Figure 5 A). In addition, anti-Flag antibody precipitated not only Flag-SIRT7 but also Myc-ALDOA, whereas anti-Myc antibody precipitated both Myc-ALDOA and Flag-SIRT7 proteins after transfection of both plasmids, highlighting the physical interaction between SIRT7 and ALDOA in cells ( Figure 5 B, C). Functionally, SIRT7 overexpression did not affect the expression of Myc-ALDOA, but reduced the K-lactylation of Myc-ALDOA ( Figure 5 D). Consistently, treatment of cells with a SIRT7 inhibitor increased K-lactylation of ALDOA, whereas treatment with resveratrol, an activator of SIRT1 and SIRT7, reduced K-lactylation of ALDOA. (Jin et al., 2023; Tang et al., 2017) (28827661, 36587031) Knockout of Myc-ALDOAK lactylation ( Figure 5E, F). In addition, the percentage of ccRCC tumors with high SIRT7 expression was significantly lower than that of NATs ( Figure 5 K).

[0172] Next, we tested whether SIRT7 could eliminate the lactoylation of ALDOA-K230 using an anti-ALDOA-K230 lactoyl-lysine antibody. Treatment with a SIRT7 inhibitor increased the level of ALDOA-K230 lactoylation ( Figure 5 H), while resveratrol treatment reduced the level of ALDOA-K230 lactylation in HEK293T cells ( Figure 5 HEK293T cells were co-transfected with plasmids expressing Myc-WT-ALDOA or Myc-ALDOA-KR mutants, as well as Flag control or Flag-SIRT7 plasmids. Immunoprecipitation experiments revealed that only transfection of the Myc-ALDOA-K230R mutant reduced the level of Myc-ALDOA lactylation, while induction of SIRT7 expression reduced the levels of Myc-WT-ALDOAK and Myc-ALDOA-K230 lactylation, but did not alter the low level of Myc-ALDOA-K230R lactylation (Figure S4, Figure 5 GJ). Thus, SIRT7 appears to be able to clear lactoylated ALDOAK230.

[0173] 6. ALDOA-K230R molecular docking and proteomic analysis revealed the role of ALDOA in tumor malignant behavior

[0174] Tumor cells tend to consume energy through glycolysis, the Warbury effect, which is independent of oxygen levels (Warburg and O., 1925; Warburg, 1956), (Warburg, 1925) https: / / doi.org / 10.1158 / jcr.1925.148, 13298683), (Hanahan and Weinberg, 2011)(21376230) However, glycolysis is less efficient in producing ATP, increasing glucose consumption and lactate content in tumor cells (Boroughs and DeBerardinis, 2015; Lunt and VanderHeiden, 2011; Vander Heiden et al., 2009)(19460998, 21985671, 25774832). ALDOA is an important enzyme in glycolysis, promoting the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Therefore, the intracellular ATP, lactate, and glucose concentrations in the supernatant of cultured ccRCC cells were assessed to investigate the role of ALDOA and ALDOA-K230 lactylation in regulating the Weinberg effect. As expected, ALDOA silencing significantly increased glucose concentration in the supernatant of cultured ccRCC 786-O cells, but reduced lactate levels ( Figure 6 A, B), while the intracellular ATP concentration decreases ( Figure 6 C). Therefore, ALDOA silencing significantly inhibited glucose consumption and glycolysis, which is consistent with previous studies. (Fu et al., 2018; Wang et al., 2021)(29764507,33436261). Forced expression of WT-ALDOANTm (silencing nucleotide mutation) or mutant ALDOAK230RNTm restored glucose consumption and glycolysis in ALDOA-silenced 786-O cells compared to ALDOA-silenced cells ( Figure 6 AC). Apparently, ALDOA-K230 lactoylation has little effect on regulating the levels of glycolytic metabolites in ccRCC cells. In addition, induction of WT-ALDOANTm or ALDOAK230RNTm almost completely restored ALDOA enzyme activity in ALDOA-silenced 786-O cells compared with control cells ( Figure 6 D). Therefore, ALDOAK230 lactoylation may not be a key factor for ALDOA enzyme activity. Consistently, previous studies have shown that ALDOAD33, Y361, Cys73, and Cys339 (Chang et al., 2020; Qin et al., 2019)(32188842,31332308) are essential for enzyme activity and glycolysis. It is hypothesized that ALDOA-K230 lactoylation may affect the molecular structure of ALDOA and enhance tumor malignant behavior by interacting with ATCG1 (Gizak et al., 2019)(31558701). Therefore, molecular docking experiments were performed and it was found that the structure of K230-lactoylated ALDOA was significantly different from that of unlactoylated ALDOA, with a root mean square deviation (RMSD) of 2.166 ( Figure 6E). Moreover, K230-lactylated ALDOA has significantly higher binding affinity to ACTG1 than un-lactylated ALDOA Figure 6 F). The PIPER potential of the lactylated ALDOA-ACTG1 complex is -1569.854, while this energy for the un-lactylated ALDOA-ACTG1 complex is -973.18 Figure 6 F). We further explored the function of ALDOA by performing proteomic analysis on ALDOA-silenced ccRCC tumor cells transfected with ALDOA-WT or ALDOA-K230R Figure 6 G-J). Induction of ALDOA-K230R mutant expression significantly reduced the levels of MYC and E2F-targeted gene targets in ccRCC cells compared to ALDOA-WT cells Figure 6 K), stemness score and CD44 expression Figure 6 L-N). These suggest that ALDOA-K230 lactylation can be crucial for the proliferation and stemness maintenance of ccRCC cells.

[0175] 7. ALDOA-K230 lactylation is important for maintaining the malignant behavior of ccRCC cells

[0176] To determine the exact role of ALDOA silencing and ALDOA-K230 lactylation in the malignant behavior of ccRCC tumor cells, ccRCC 786-O cells stably expressing ALDOA silencing (shALDOA) were constructed using shRNA-based lentivirus Figure 7 A). The ALDOA silencing efficiency was highest in cells stably expressing 126867 ALDOA shRNA, which almost reduced ALDOA expression by 70%. Therefore, this cell line was selected for subsequent experiments. Functionally, ALDOA silencing significantly reduced the relative expression levels of vimentin, fibronectin and N-cadherin in 786-O cells, indicating that ALDOA silencing inhibited the EMT process of 786-O cells, which is consistent with previous research results. (Ji et al., 2016) (26854714) Figure 7 B). Introduction of WT-ALDOA NTm, but not ALDOA-K230R NTm expression, restored the expression levels of vimentin, fibronectin and N-cadherin in ALDOA-silenced 786-O cells, highlighting the importance of ALDOA-K230 lactylation in the EMT process of 786-O cells Figure 7C). In addition, while ALDOA silencing significantly inhibited the dynamic proliferation of 786-0 cells, the introduction of Myc-WT-ALDOA expression completely restored the proliferation of 786-0 cells, while the introduction of Myc-ALDOA-K230R mutant expression only partially restored the proliferation of 786-0 cells. Similar data patterns were also obtained in wound healing, invasion and apoptosis experiments, but the introduction of ALDOA-K230R mutant expression did not significantly restore the clonogenic capacity of 786-0 cells Figure 7 D-G, J). In addition, ALDOA silencing significantly increased the levels of Cleaved-Caspase3 and Cleaved-PARP1 in 786-0 cells Figure 7 H), which is similar to previously reported results. (Saito et al., 2020) (32530543). In ALDOA-silenced 786-0 cells, the restoration of WT-ALDOA NTm significantly alleviated the increased expression of apoptosis markers, while the expression of ALDOA-K230R NTm did not have this effect Figure 7 I). These data suggest that ALDOA can enhance the malignant behavior of ccRCC cells as an oncogenic factor, partially dependent on its K230 site lactylation.

[0177] III. Conclusion

[0178] Integrative multi-omics analyses, including proteomics, phosphoproteomics, and glycoproteomics, revealed novel molecular mechanisms underlying the pathogenesis of ccRCC in this study. Analysis of proteomics, phosphoproteomics, and phosphotylomics revealed that differentially expressed and phosphorylated proteins with varying levels of K-phosphotyloylation in ccRCC tissues were involved in enhanced HIF signaling, glycolysis, DNA replication, and immune-related pathways, while attenuating glucose oxidative phosphorylation and amino acid and fatty acid metabolism, consistent with previous observations (Clark et al., 2019; Hu et al., 2024a; Qu et al., 2022) (31675502,3544054238361033). A regulatory network was constructed in ccRCC based on the proteome, phosphoproteomics, kinase activity, and transcription factor activity, forming two core oncogenic subnetworks: NFKB1-RELA-SP1-TP53 and MAPK1-MAPK3-MAPK14-JUN. These findings support the concept that NFKB promotes tumor malignancy and modulates its immune microenvironment, contributing to tumor immune evasion. (Erez et al., 2010)[20138012] Indeed, upregulated NFKB expression and enhanced NFKB signaling are often associated with poor prognosis and lower survival in ccRCC patients (Peri et al., 2013)[24116146], highlighting the potential of targeting NFKB as a therapeutic strategy for ccRCC. Our study also indicates that key events in the MAPK pathway, such as MAPK1, MAPK3, and MAPK14, are major drivers of ccRCC development. A panel of MAPK signature genes serves as biomarkers for prognosis and assessment of treatment response in ccRCC patients, and several key MAPK signaling events are independent risk factors for poor prognosis in ccRCC because they support the malignant behavior of ccRCC. (Zheng et al., 2024)[39038636] It can be imagined that simultaneously targeting the NFKB and MAPK signaling pathways may be of great value in the intervention of ccRCC, so our findings may provide a new therapeutic target for the intervention of ccRCC.

[0179] Recent studies have shown that K-lactylation of histones and non-histone proteins is associated with altered transcription factor activity and enhances the malignant behavior of tumors by activating related signals (Hu et al., 2024b)(39522689). K-lactylation in histones promotes the progression of ccRCC by enhancing PDGFRβ signaling (Yang et al., 2022)[35637958]. It was observed that the number of histone Kla sites was generally increased in ccRCC tumors compared with NATs ( Figure 3B) Similarly, increased Kla sites in histones are enriched for proliferation-related pathways. Clearly, not only are Kla sites increased in non-histone proteins, but Kla sites in histones also contribute to the malignant behavior of ccRCC. Therefore, the precise function of increased Kla sites likely depends on the nature of the protein and the microenvironment.

[0180] Analysis of the non-histone protein Kla revealed the presence of 11 proteins containing >10 Kla sites in ccRCC tumors, including the glycolytic enzymes ALDOA, ENO1, PGK1, and PKM. Interestingly, upregulation of ALDOA expression was detected in ccRCC tissues and correlated with poor survival in ccRCC patients. (Gizak et al., 2019)(31558701) ALDOA is a conserved protein in different species and may be a good druggable target (Feng et al., 2024)[39099416] The protein contains two domains of amino acids 1-14 and amino acids 15-363, the latter of which is crucial for converting fructose 1,6-bisphosphate (F1,6BP) to 3-phosphoglycerate (G3P) and dihydroxyacetone phosphate (DHAP), promoting glycolytic flux and lactate production (Chang et al., 2018).[29907340](Arakaki et al., 2004)[15537755] In addition, lactylation of ALDOA was detected in ccRCC tissues and HEK293T cells ( Figure 4 A, C, D), and high-concentration lactate treatment of 786-O cells significantly enhanced this process. In fact, lactate levels in ccRCC tissues were significantly higher than those in NATs ( Figure 4 A). More importantly, we found that the lactoyltransferase CBP acts as the "writer" of ALDOA and ALDOA-K230 lactylation ( Figure 4 EQ), while the deacetylase SIRT7 acts as an eraser of ALDOA and ALDOA-K230 lactylation ( Figure 5 As expected, ALDOA silencing significantly reduced glycolysis and decreased ATP and lactate production ( Figure 6 AD), which is consistent with previous studies. (Fu et al., 2018; Wang et al., 2021) [29764507,33436261] Unexpectedly, it was found that lactoylation of ALDOA-K230 did not regulate its enzymatic activity and glycolysis in ccRCC cells. Apparently, reintroduction of ALDOA-K230R mutant expression, similar to wild-type ALDOA, almost completely restored the enzymatic activity and glycolysis of ALDOA in silenced 786-O cells ( Figure 6AD). These unexpected data suggest that in non-histone proteins, Kla does not always change the original function of the protein. Interestingly, molecular docking showed that ALDOA, especially K230 lactylated ALDOA, was predicted to interact with ACTG1, a γ-actin that activates the Ras / MAPK signaling pathway, promoting invasive proliferation, clonogenicity, invasion, and resistance to spontaneous apoptosis and drug treatment. (Xiao et al., 2021)(34767732). Previous studies have shown that ALDOA promotes tumor cell proliferation, invasion, and anti-apoptosis by interacting with ATCG1 (Gizak et al., 2019)[31558701] Consistently, proteomic analysis showed that reintroduction of ALDOA-K230R mutant expression impaired the expression of proliferation-related markers (MYC and E2F targets) ( Figure 6 K), reduced cancer stem cell score and CD44 expression in ALDOA-silenced 786-O cells ( Figure 6 LN). In addition, reintroduction of the ALDOA-K230R mutation failed to fully restore the epithelial-mesenchymal transition process and only partially restored the proliferation, clonogenicity, invasiveness and spontaneous apoptosis of ALDOA-silenced 786-O cells. ALDOA-K230 emulsifies by interacting with ACTG1, activating the RAS / MAPK signaling pathway and its "moonlighting function", which may enhance the malignant behavior of ccRCC cells. The data of this application extend previous observations that ALDOA promotes tumorigenesis and progression of non-small cell lung cancer cells by activating the EGFR / MAPK pathway (Fu et al., 2018)[29764507] and promotes the proliferation and invasion of cholangiocarcinoma cells (Li et al., 2021)[33994862]. The new findings of this application suggest that ALDOA-K230 emulsification may be a therapeutic target for intervention in ccRCC.

[0181] Lactoylation profiling revealed that in ccRCC tissues, an increase in the number of Kla sites in histones was associated with the expression of several acyltransferases and deacylases, but not CBP and SIRT7. However, CBP and SIRT7 were found to act as writers and erasers, respectively, of ALDOA-K230 lactoylation. These data suggest that certain important writers and erasers may be expressed at moderate levels, but their functions are essential for the balance of Kla sites in specific proteins. Alternatively, these specific acyltransferases and deacylases may be associated with an increase in the number of Kla sites in non-histone proteins. Therefore, when identifying specific writers and erasers, acyltransferases and deacylases with altered expression levels and Kla site numbers in non-histone proteins should be considered.

[0182] In summary, multi-omics analysis revealed differentially expressed and phosphorylated proteins, leading to the construction of a regulatory network. Lactoylation profiling revealed that ALDOA, a key enzyme in glycolysis, is upregulated in ccRCC, and that ALDOA-K230 lactoylation is mediated by CBP and abrogated by SIRT7. K230 lactoylation of ALDOA is not essential for glycolysis but is crucial for the malignant behavior of ccRCC cells. Therefore, ALDOA-K230 lactoylation may represent a novel target for intervention in ccRCC.

Claims

1. A kit for detecting clear cell renal cell carcinoma, the kit comprising: a) at least one agent capable of inhibiting lactylation modification at K230 of aldolase A (ALDOA); b) at least one agent capable of modulating the activity of histone acetyltransferase CBP (CREB binding protein); and / or c) at least one agent capable of modulating the activity of the sirtuin SIRT7.

2. The detection kit according to claim 1, wherein The agent capable of regulating the activity of histone acetyltransferase CBP (CREB binding protein) refers to an agent capable of downregulating the activity of histone acetyltransferase CBP (CREB binding protein).

3. The detection kit according to claim 1 or 2, wherein The reagent capable of regulating the activity of the deacetylase SIRT7 refers to a reagent capable of upregulating the activity of the deacetylase SIRT7.

4. A detection kit for identifying the malignancy of clear cell renal cell carcinoma, the kit comprising a reagent for detecting the lactylation modification level of the K230 site of aldolase A (ALDOA).

5. The detection kit according to claim 4, wherein The kit is equipped with a lactic acidification level grading standard: If the positive rate of immunohistochemical staining is less than 30%, it is considered to be of low malignancy; If the positive rate of immunohistochemical staining is 30%-60%, it is considered to be of moderate malignancy; If the positive rate of immunohistochemical staining is greater than 60%, it is considered to be highly malignant.

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