Application of TGM2 to regulation and control of ubiquitination degradation of KRAS through non-classical enzyme activity pathway and prevention and treatment of related cancers

Abstract

The invention discloses application of TGM2 to regulation and control of ubiquitination degradation of KRAS through a non-classical enzyme activity pathway and prevention and treatment of related cancers, and belongs to the technical field of biological medicines. It is verified that TGM2 inhibits KRAS ubiquitination through a non-classical enzyme activity pathway, key ubiquitination sites on KRAS are determined, and the structural basis of interaction of TGM2 and KRAS is clarified. The inhibiting effect of the TGM2 inhibitor on proliferation and metastasis of KRAS related cancer cells is screened and verified, the synergistic effect of the TGM2 inhibitor and chemotherapeutic drugs is evaluated, and a new strategy is provided for clinical treatment. The correlation between the TGM2 expression level and the prognosis of the KRAS-related cancer patient is confirmed through related experiments, and a convenient and efficient TGM2 detection method is developed and used for patient stratification and treatment scheme optimization. The technical problems that existing KRAS targeted therapy is narrow in application range and inaccurate in prognosis evaluation are solved, and a brand new scheme is provided for accurate diagnosis and treatment of KRAS related cancers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of TGM2 in regulating KRAS ubiquitination degradation and preventing and treating related cancers through non-classical enzymatic pathways. Background Technology

[0002] KRAS (Kirsten rat sarcoma virus oncogene homolog) is a member of the RAS family. As the most common mutated oncogene in human cancers, its mutations occur frequently in various malignant tumors such as pancreatic cancer (approximately 85%), lung cancer (approximately 31%), and colorectal cancer (approximately 43%) (Trends Cancer. 2024 Dec 27; PMID: 39732595). As a GTPase, KRAS protein exists in cells in an inactive state bound by GD and an active state bound by GTP. Mutated KRAS (such as G12D, G12V, and G12C) loses its GTPase activity and remains in an active state. By activating downstream signaling pathways such as PI3K / AKT and RAS / RAF / MEK / ERK, it promotes tumor cell proliferation, survival, migration, and metabolic reprogramming (Nature. 2019 Mar 13; 567(7749): 535-539).

[0003] For a long time, the treatment of KRAS-mutant cancers has faced enormous challenges: on the one hand, the KRAS protein itself lacks a binding pocket for traditional small molecule drugs and is considered an "untreatable" target; on the other hand, existing drugs targeting downstream KRAS pathways (such as MEK inhibitors and PI3K inhibitors) are prone to drug resistance and are highly toxic to normal cells (Science. 2024 Jun 7;384(6700):eadk0775). In recent years, although specific inhibitors targeting KRAS G12C mutations (such as Sotorasib and Adagrasib) have been approved for marketing, they are only applicable to patients with G12C mutations (accounting for about 13% of KRAS mutations), limiting the applicable population, and drug resistance issues still exist (Nature. 2024 May;629(8013):927-936). Therefore, finding novel mechanisms regulating KRAS protein stability and developing broad-spectrum treatment strategies applicable to multiple KRAS mutation types have become core needs of current cancer research.

[0004] The transglutaminase (TGM) family is a class of calcium-dependent enzymes comprising nine members (TGM1-TGM7, F13A1, EPB4.2). Their classic function is to catalyze the formation of heteropeptide bonds between glutamine and lysine residues in proteins, participating in physiological processes such as apoptosis and extracellular matrix formation (Physiol Rev. 2014 Apr). Recent studies have found that TGM family members (especially TGM2) are highly expressed in various cancers and are closely related to tumorigenesis and development—for example, TGM2 is highly expressed in pancreatic cancer, enhancing tumor metastasis by promoting EMT; in lung cancer, TGM2 promotes tumor proliferation by activating the PI3K / AKT signaling pathway (Oncogene. 2021 Jun;40(25):4352-4367). However, the direct association between the TGM family and the KRAS signaling pathway has not been reported, and whether it affects cancer progression by regulating KRAS protein stability remains an unknown area.

[0005] The ubiquitin-proteasome system (UPS) is a major pathway for intracellular protein degradation. KRAS protein ubiquitination and degradation are jointly regulated by E3 ubiquitin ligases (such as WWP1 and ITCH) and deubiquitinating enzymes (such as USP7 and OTUB1) (Cancer Res. 2020 Mar 24; PMID: 32209560). Existing research indicates that downregulating KRAS ubiquitination levels leads to KRAS protein accumulation and enhances its oncogenic activity; conversely, promoting KRAS ubiquitination and degradation can inhibit tumor progression. Therefore, identifying key molecules regulating KRAS ubiquitination has become an important direction for developing KRAS-related cancer therapeutics. However, there are currently no reports of TGM family members participating in the regulation of KRAS ubiquitination, and their potential mechanisms urgently need to be explored.

[0006] In summary, the existing technologies generally have the following problems: (1) KRAS targeted therapy has a narrow scope of application and significant drug resistance: Existing KRAS inhibitors (such as Sotorasib) only target G12C mutations and cannot cover high-frequency mutation types such as G12D, G12V, G13D, and Q61H; and long-term use is prone to KRAS secondary mutations (such as G12C→G12D) or downstream pathway activation (such as BRAF mutations), resulting in drug resistance and limited therapeutic effects. (2) The KRAS ubiquitination regulation mechanism is unclear and there is a lack of effective intervention targets: Although it is known that KRAS ubiquitination is regulated by E3 ubiquitin ligases and deubiquitinating enzymes, existing studies have mostly focused on single enzyme molecules and have not found key molecules that can broadly regulate KRAS ubiquitination; and some regulatory molecules (WWP1) are also highly expressed in normal cells, and targeted inhibition is prone to side effects. (3) The mechanism of action of the TGM family in cancer is incomplete: Existing studies have only confirmed that TGM2 affects cancer progression by promoting EMT or activating the PI3K / AKT pathway, but whether it directly interacts with key KRAS oncogenes or regulates cancer-related signaling pathways through non-classical enzymatic pathways is still unclear, which limits the development of TGM as a cancer therapeutic target. (4) KRAS-related cancers lack effective prognostic biomarkers: Existing prognostic assessment indicators (such as tumor stage and pathological grade) are difficult to accurately reflect the differences in patient prognosis; although some studies have reported that KRAS mutation type is related to prognosis, the detection cost is high and it cannot guide the selection of treatment plans, thus limiting its clinical application. Summary of the Invention

[0007] Based on the aforementioned deficiencies in the existing technology, the team of this invention discovered through previous research that TGM2 is highly expressed in KRAS-related cancers and is positively correlated with the expression level of KRAS. Further experiments confirmed that TGM2 can directly interact with KRAS, inhibiting KRAS ubiquitination and degradation through non-classical enzymatic pathways, leading to the accumulation of KRAS protein and enhancing its carcinogenic activity. Based on this discovery, the research and development concept of this invention is as follows: (1) Clarify the mechanism by which TGM2 regulates KRAS ubiquitination: Through mutant experiments, ubiquitination detection and other methods, verify whether TGM2 inhibits KRAS ubiquitination through non-classical enzymatic pathways (independent of classical transglutaminase activity), identify key ubiquitination sites on KRAS (such as K117), and elucidate the structural basis of their interaction. (2) Develop TGM2 inhibitors for the treatment of KRAS-related cancers: Based on the regulatory role of TGM2 in KRAS ubiquitination, screen and verify the inhibitory effects of TGM2 inhibitors (such as Cystamine and GK921) on the proliferation and metastasis of KRAS-related cancer cells, evaluate their synergistic effect with chemotherapy drugs, and provide new strategies for clinical treatment. (3) Establish a detection method for TGM2 as a prognostic biomarker for KRAS-related cancers: Through database analysis and clinical sample verification, confirm the correlation between TGM2 expression level and the prognosis of patients with KRAS-related cancers, and develop convenient and efficient TGM2 detection methods (such as immunohistochemistry and qPCR) for patient stratification and treatment optimization. Therefore, the purpose of this invention is to provide the application of TGM2 in regulating KRAS ubiquitination degradation through non-classical enzymatic pathways, develop the use of TGM2 inhibitors in the treatment of KRAS-related cancers, and establish a detection method for TGM2 as a prognostic biomarker to solve the technical problems of narrow applicability and inaccurate prognostic assessment of existing KRAS-targeted therapies, and provide new solutions for the precision diagnosis and treatment of KRAS-related cancers.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide the application of TGM2 inhibitors in the preparation of KRAS ubiquitination-regulating drugs.

[0009] Furthermore, the regulation is positive, increasing the level of KRAS ubiquitination.

[0010] Furthermore, the TGM2 inhibitors include Cystamine and GK921.

[0011] Furthermore, the drug also includes one or more pharmaceutically acceptable carriers.

[0012] The second objective of this invention is to provide the application of TGM2 inhibitors in the preparation of drugs for the prevention and treatment of KRAS-related cancers.

[0013] Furthermore, KRAS gene mutations are frequently expressed in the aforementioned KRAS-related cancers.

[0014] Furthermore, the TGM2 inhibitor prevents and treats KRAS-related cancers by increasing KRAS ubiquitination levels.

[0015] Furthermore, the TGM2 inhibitors include Cystamine and GK921.

[0016] Furthermore, the drug also includes one or more pharmaceutically acceptable carriers.

[0017] The third objective of this invention is to provide the application of TGM2 detection reagent in the preparation of KRAS-related cancer prognostic assessment products.

[0018] Compared with the prior art, the present invention has the following core advantages, and the relationship between each advantage and the corresponding technical point and the reasons for their occurrence are as follows: 1. Precisely targets and regulates KRAS stability, overcoming the limitations of traditional treatments. In existing technologies, KRAS-mutant cancers (such as pancreatic cancer, colorectal cancer, and lung cancer) have long faced the dilemma of being "untreatable" due to the lack of drug-binding pockets for the KRAS protein itself. This invention discovers that TGM2 can inhibit the ubiquitination and degradation of KRAS through a non-classical enzymatic pathway, thereby maintaining high KRAS protein expression—a mechanism that provides a novel target for the treatment of KRAS-related cancers. By regulating TGM2 activity (e.g., using the TGM2-specific inhibitor GK921), KRAS protein levels can be directly reduced, and this is effective for both wild-type (WT) and mutant KRAS (such as G12D, G12V, G12C, G13D, Q61H, etc.), solving the problem that traditional drugs only target specific KRAS mutation sites (such as G12C) and have limited applicability to certain populations. This advantage arises because TGM2 does not depend on its canonical transglutaminase activity (the C277 mutation does not affect its regulatory role on KRAS), but rather by directly interacting with KRAS, inhibiting KRAS ubiquitination levels, reducing KRAS degradation via the ubiquitin-proteasome pathway, and thus maintaining KRAS protein stability. When TGM2 inhibitors are used or TGM2 expression is knocked down, KRAS ubiquitination levels increase, protein degradation accelerates, thereby inhibiting KRAS signaling pathway activation.

[0019] 2. TGM2 inhibitors have both anti-proliferation and anti-metastasis effects, resulting in a more comprehensive therapeutic effect. Existing KRAS-related cancer treatments primarily focus on inhibiting tumor cell proliferation, with insufficient intervention in tumor metastasis, a critical link leading to patient death. The TGM2 inhibitors in this invention (such as the pan-TGM inhibitor Cystamine and the TGM2-specific inhibitor GK921) not only inhibit tumor cell proliferation by reducing KRAS levels but also significantly inhibit tumor cell metastasis by downregulating the expression of the transcription factor Slug and suppressing the expression of tumor cell epithelial-mesenchymal transition (EMT)-related proteins (such as N-CAD and VIME). This is because, on the one hand, TGM2 promotes tumor cell proliferation by stabilizing KRAS and activating the PI3K / AKT signaling pathway; on the other hand, TGM2 enhances tumor cell migration and invasion by upregulating Slug expression and inducing EMT. TGM2 inhibitors can simultaneously block these two pathways, achieving a dual therapeutic effect of "proliferation inhibition + metastasis blocking."

[0020] 3. TGM2 as a prognostic biomarker aids in precision cancer diagnosis and treatment. In existing technologies, there is a lack of effective prognostic biomarkers for KRAS-related cancers, making it difficult to guide the selection of clinical treatment plans. This invention, through analysis of databases such as GEPIA and TCGA, found that high TGM2 expression is significantly associated with poor prognosis in patients with KRAS-related cancers (e.g., in PAAD patients, the overall survival of patients with high TGM2 expression was significantly shorter than that of patients with low expression, p=0.007; in LGG patients, p=0.029; and in LUSC patients, p=0.011), and TGM2 expression levels are positively correlated with KRAS expression levels (R values ​​were 0.59, 0.63, and 0.57, respectively, all p<0.001). TGM2 stabilizes KRAS protein, causing tumor cells to remain in a state of malignant proliferation, leading to a worse prognosis. Simultaneously, single-cell sequencing and spatial transcriptome data show that TGM2 is highly expressed in tumor tissues (especially pancreatic tumor cells) but expressed at low levels in normal tissues. This characteristic makes it a specific biomarker for prognostic assessment of KRAS-related cancers, which can be used for patient stratification and treatment optimization.

[0021] 4. TGM2 inhibitors have high safety profiles and reduce off-target effects. Existing non-specific KRAS inhibitors easily affect the KRAS signaling pathway in normal cells (KRAS is involved in the regulation of growth and development in normal cells), leading to serious side effects. The TGM2 inhibitors in this invention (such as GK921) target only tumor cells with high TGM2 expression—because the expression level of TGM2 is low in normal tissues, the inhibitors have less impact on the KRAS signaling pathway in normal cells, significantly reducing off-target effects. Single-cell sequencing data in this invention show that the expression level of TGM2 in pancreatic tumor tissue is significantly higher than that in normal pancreatic tissue (p<2e-16), and it is mainly expressed in tumor cells, while its expression is extremely low in acinar cells and ductal cells of normal pancreas; spatial transcriptome data further confirms that TGM2 is enriched in the tumor region and peritumoral infiltration area, and almost not expressed in normal areas. This tissue-specific expression allows the TGM2 inhibitors to precisely target tumor cells, reducing damage to normal tissues. Attached Figure Description

[0022] Figure 1 This is the result of a multi-omics analysis of the TGM family and pancreatic cancer, and an exploration of TGM2 function, in Example 1 of this invention.

[0023] Figure 2 This illustrates the effect of the TGM2 inhibitor Cystamine and GK921 on the inhibition of pancreatic cancer cell proliferation in Example 2 of the present invention.

[0024] Figure 3 This describes the inhibition of pancreatic cancer cell metastasis by the TGM2 inhibitor Cystamine and GK921 in Example 3 of the present invention.

[0025] Figure 4 This is the result of the study on the regulation of Ras protein expression by the TGM family and its interaction with KRAS in Example 4 of the present invention.

[0026] Figure 5 This describes the situation in Example 5 of the present invention where TGM2 regulates KRAS ubiquitination degradation through a non-classical enzymatic pathway. Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0028] This invention provides the following technical solutions 1. TGM2 inhibitor products The TGM2 inhibitors involved in this invention include pan-TGM inhibitors and TGM2-specific inhibitors. The specific product composition, structure and physical properties are shown in Table 1.

[0029] Table 1. Product Details of TGM2 Inhibitors

[0030] 2. Reagents for TGM2 detection It is used to detect the expression level of TGM2 in biological samples, and to assist in the prognostic assessment and treatment selection of KRAS-related cancers. The product composition is shown in Table 2.

[0031] Table 2. Details of TGM2 detection reagents.

[0032] The detection range of this protocol is: TGM2 protein concentration 0.1-10 μg / mL (Western Blot); immunohistochemistry can detect the expression and localization of TGM2 in tissue samples, and the interpretation criteria are "positive cell ratio ≥50% is high expression, <50% is low expression".

[0033] The method of the present invention is as follows: I. Methods for TGM2 inhibitors to inhibit the proliferation of KRAS-related cancer cells This method involves treating KRAS-related cancer cells with a TGM2 inhibitor to reduce KRAS protein levels and inhibit cell proliferation. The specific steps are as follows: [1] Cell preparation 1. Select KRAS-related cancer cell lines, including but not limited to: pancreatic cancer Capan-1 cells (KRASG12D mutation), Capan-2 cells (KRASG12V mutation), BXPC-3 cells (KRASWT), and PANC-1 cells (KRASG12D mutation).

[0034] 2. Cell culture conditions: Use DMEM or RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep) and culture at 37°C in a 5% CO2 incubator until the logarithmic growth phase (cell density reaches 5×10⁶ cells / year). 4 -1×10 5 (cells / mL).

[0035] [2] Inhibitor treatment 1. Preparation of inhibitor stock solution: Dissolve Cystamine (pan-TGM inhibitor) in physiological saline to prepare a stock solution with a concentration of 100 mM; dissolve GK921 (TGM2 specific inhibitor) in DMSO to prepare a stock solution with a concentration of 10 mM, and store at -20℃.

[0036] 2. Serial dilution: Dilute the stock solution with complete culture medium to the working concentration. The working concentration range of Cystamine is 0.1-2 mM (optimal concentration is 0.5 mM), and the working concentration range of GK921 is 0.25-5 μM (optimal concentration is 1 μM). At the same time, set up a control group (add only an equal volume of physiological saline or DMSO, with a final concentration of DMSO ≤0.1% to avoid cytotoxicity).

[0037] 3. Processing steps: Seed cells in the logarithmic growth phase into 96-well plates (1 × 10⁶ cells per well). 3 -5×10 3 After adhering to the wells for 24 hours, the old culture medium was discarded and fresh culture medium containing different concentrations of inhibitors was added. Three replicates were set up for each group, and the culture was continued for 24-72 hours (the optimal culture time is 48 hours).

[0038] (1) Cell proliferation was detected by CCK-8 method: After the culture was completed, 10 μL of CCK-8 reagent was added to each well and incubated at 37℃ for 2 h. The absorbance (OD value) was measured at 450 nm using an ELISA reader.

[0039] (2) Calculate the proliferation inhibition rate: Proliferation inhibition rate = (1 - OD value of experimental group / OD value of control group) × 100%, plot the inhibitor concentration-inhibition rate curve, and calculate IC50. 50 (Half-maximal inhibitory concentration).

[0040] [3] Result verification 1. Western Blot detection of KRAS protein levels: Cells treated with inhibitors were collected, total protein was extracted, protein concentration was measured, and SDS-PAGE electrophoresis was performed. The protein was then transferred to a PVDF membrane, incubated with anti-KRAS antibody (1:1000 dilution), anti-β-Actin antibody (1:5000 dilution), and HRP-labeled secondary antibody (1:2000 dilution). After ECL staining, the relative expression level of KRAS protein was quantitatively analyzed using ImageJ software.

[0041] 2. Clonogenic assay verification: Inhibitor-treated cells were seeded into 6-well plates (100-200 cells per well), cultured for 10-14 days, fixed with methanol, stained with crystal violet, and the number of clones was counted (clones with ≥50 cells were considered valid clones). The clonal formation rate was calculated as (number of clones in the experimental group / number of clones in the control group) × 100%.

[0042] II. Methods for TGM2 inhibitors to inhibit KRAS-related cancer cell metastasis This method involves treating KRAS-related cancer cells with a TGM2 inhibitor to downregulate Slug expression and inhibit the EMT process. The specific steps are as follows: [1] Cell preparation 1. Select KRAS-related cancer cell lines with high metastatic potential, such as Capan-1 (KRASG12D), Capan-2 (KRASG12V), BXPC-3 (KRASWT), and lung cancer H1299 (KRASWT) cells.

[0043] 2. Cell culture conditions are the same as in 3.2.1. After culturing to the logarithmic growth phase, the cells are starved with serum-free medium for 12 hours to synchronize the cell cycle.

[0044] [2] Inhibitor treatment 1. The inhibitor concentration was set as in 3.2.1 (Cystamine 0.1-2mM, GK921 0.25-5μM). The control group was treated with an equal volume of physiological saline or DMSO for 24-48 hours (the optimal time was 36 hours).

[0045] 2. Transfer capacity testing Transwell migration assay: The treated cells were resuspended in serum-free medium to a concentration of 1×10⁻⁶. 5 Cells / mL: 200 μL of cell suspension was added to the upper chamber of a Transwell apparatus, and 600 μL of medium containing 10% FBS was added to the lower chamber. The mixture was incubated at 37°C for 24 h. After methanol fixation and crystal violet staining, unmigrated cells in the upper chamber were wiped away with cotton swabs. The number of migrating cells in the lower chamber was counted under a microscope. The migration inhibition rate was calculated as (1 - number of migrating cells in the experimental group / number of migrating cells in the control group) × 100%.

[0046] Scratch healing assay: The treated cells were seeded into 6-well plates and cultured until the confluence reached 90%. A straight line was drawn on the bottom of the well using a 200 μL pipette tip. The exfoliated cells were washed away with PBS, and serum-free medium containing inhibitors was added. The cells were photographed at 0h, 24h, and 48h. The scratch width was measured using ImageJ software, and the healing rate was calculated as (0h scratch width - experimental group scratch width) / 0h scratch width × 100%.

[0047] [3] Mechanism verification Western blotting detection of EMT-related proteins: Total protein was extracted from cells treated with inhibitors and detected by anti-Slug antibody (1:1000), anti-N-CAD antibody (1:1000), anti-VIME antibody (1:1000), anti-ZO1 antibody (1:1000), and anti-β-Actin antibody (1:5000) to quantitatively analyze changes in protein expression.

[0048] III. Verification Method for TGM2 Inhibiting KRAS Ubiquitination and Degradation via Non-Classical Enzymatic Pathways This method was used to verify the mechanism by which TGM2 inhibits KRAS ubiquitination and degradation through a non-canonical enzymatic pathway. The specific steps are as follows: [1] Plasmid construction and transfection 1. Construction of recombinant plasmids: including Flag-TGM2 wild-type (WT) plasmid, Flag-TGM2 mutant plasmid (C277S, classic transglutaminase active site mutation; R580A, transglutaminase activity enhancement mutation; S171E, GTPase activity mutation), HA-KRAS wild-type (WT) plasmid, HA-KRAS mutant plasmid (K42R, K117R, K147R, K170R and other ubiquitination site mutations), His-Ub (histidine-tagged ubiquitin) plasmid.

[0049] 2. Cell transfection: Seed 293T cells or Capan-1 cells into 6-well plates and culture until the cell density reaches 70%-80%. Use Lip8000 transfection reagent to transfect the above plasmids into the cells according to the instructions. Collect cells for subsequent experiments 48 hours after transfection.

[0050] [2] Co-immunoprecipitation (Co-IP) was used to verify the interaction between TGM2 and KRAS. 1. Cell lysis: Transfected cells were lysed with pre-cooled RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors), incubated at 4°C for 30 min, centrifuged at 12,000 rpm for 15 min, and the supernatant (total protein) was collected.

[0051] 2. Immunoprecipitation: Take 500 μg of total protein, add 2 μg of anti-Flag antibody (for detecting Flag-TGM2) or anti-HA antibody (for detecting HA-KRAS), incubate overnight at 4°C, add Protein A / G agarose beads, incubate at 4°C for 2 h, centrifuge to collect the agarose beads, wash 3 times with pre-cooled PBS, add 1×SDS loading buffer, boil for 5 min, centrifuge to collect the supernatant.

[0052] 3. Western Blot detection: The supernatant was subjected to SDS-PAGE electrophoresis, and after transfer to a membrane, it was incubated with anti-HA antibody (to detect HA-KRAS) or anti-Flag antibody (to detect Flag-TGM2), followed by ECL color development to verify whether there is an interaction between TGM2 and KRAS; at the same time, the interaction between TGM2 mutants (such as C277S, R580A) and KRAS was detected to determine whether the classical enzyme active site affects the binding of the two.

[0053] [3] Ubiquitination assay to detect KRAS ubiquitination level 1. Cell treatment: While transfecting HA-KRAS and His-Ub plasmids, transfect Flag-TGM2 WT or mutant plasmids, or add TGM2 inhibitors (Cystamine 0.5mM, GK921 1μM) and treat for 48h; to inhibit proteasome degradation, MG132 (10μM) can be added 6h before cell collection.

[0054] 2. Ubiquitination detection: Nickel column affinity purification (His-pulldown): After cell lysis, 1 mg of total protein was taken and added to nickel-NTA agarose beads. The mixture was incubated at 4°C for 4 h. After washing, 2×SDS loading buffer (containing β-mercaptoethanol) was added. After boiling, Western blotting was performed. The ubiquitination level of HA-KRAS was detected using anti-HA antibody (ubiquitinated KRAS appeared as a band with a higher molecular weight).

[0055] Co-IP assay: HA-KRAS was immunoprecipitated with anti-HA antibody, and Western blotting was performed using anti-Ub antibody (to detect ubiquitin) or anti-His antibody (to detect His-Ub) to quantitatively analyze changes in KRAS ubiquitination levels and determine the influence of TGM2 and its mutants on KRAS ubiquitination; at the same time, the ubiquitination level of KRAS ubiquitination site mutants (such as K117R) was detected to identify the key ubiquitination sites of TGM2 action.

[0056] [4] Verification of TGM2 enzyme activity 1. Transglutaminase activity assay: The transglutaminase activity of Flag-TGM2WT and mutants (C277S, R580A) was detected using a transglutaminase activity assay kit. The reaction system contained enzyme solution, substrate (such as CBZ-Gln-Gly), and chromogenic reagent. After incubation at 37℃ for 30 min, the OD value at 405 nm was measured, and the enzyme activity units (μU / μL) were calculated to verify the changes in enzyme activity of mutants.

[0057] 2. GTPase activity assay: The GTPase activity of Flag-TGM2 WT and S171E mutants was detected using a GTPase activity assay kit. The enzyme activity was calculated by measuring the Pi content produced by GTP hydrolysis, and it was determined whether the GTPase activity affected the regulatory role of TGM2 in KRAS ubiquitination.

[0058] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0059] Example 1: Multi-omics analysis of the TGM family and pancreatic cancer and exploration of TGM2 function. [1] Data sources and analysis tools Databases: GEPIA database, TCGA database, and single-cell sequencing databases containing pancreatic tumors and normal cells.

[0060] Analysis tools: GEPIA online analysis tool, survival analysis plotting tool (for Kaplan-Meier curve plotting), single-cell data analysis platform (for cell subpopulation expression, spatial distribution, and violin plot analysis).

[0061] [2] Analysis steps 1. GEPIA Database Analysis: Comprehensive Expression Analysis of the TGM Family: Median expression levels of the TGM family (TGM1, TGM2, TGM3, etc.) in tumor (T) and normal (N) samples were extracted and a heatmap was plotted; the distribution of transcripts per million (TPM) of TGM family members in pancreatic cancer (PAAD) was analyzed and a box plot was plotted; protein expression differences were observed in conjunction with immunohistochemical staining. Correlation Analysis of TGM2 with RAS: Expression data of TGM2 with KRAS, NRAS, and HRAS were extracted and a correlation scatter plot was plotted (Pearson correlation coefficient R and P value were calculated); Overall survival analysis was performed on the high / low expression groups of TGM2 in lung squamous cell carcinoma (LUSC), low-grade glioma (LGG), and pancreatic cancer (PAAD), and Kaplan-Meier curves were plotted.

[0062] 2. TCGA Database Analysis: Prognostic Analysis of the TGM Family in Pancreatic Cancer: Expression data of TGM1, TGM2, and other family members were extracted from pancreatic cancer samples and divided into high / low expression groups. Overall survival (OS) and progression-free survival (PFS) were analyzed, Kaplan-Meier curves were plotted, and p-values ​​were calculated. Prognostic Relationship between TGM2 and KRAS Mutation: Based on KRAS mutation status (KRAS^MUT, KRAS^WT) and TGM2 expression levels (high / low), OS and PFS were analyzed for each group, Kaplan-Meier curves were plotted, and p-values ​​were calculated.

[0063] 3. Single-cell data analysis: Cell subpopulation expression analysis: Extract pancreatic single-cell data to analyze the percentage and average expression of TGM2 in different cell subpopulations such as tumor cells and stellate cells. Spatial distribution and differential analysis: Combined with tissue sections, draw spatial distribution heatmaps of TGM2 in different regions such as Mal, Bdy, and nMal; draw violin diagrams to analyze the differences in TGM2 expression in different regions and their statistical significance (P-value).

[0064] [3] Analysis Results 1. Overall expression of the TGM family in the GEPIA database: Median expression of different members of the TGM family differed between tumor and normal samples; box plots and immunohistochemical staining showed that the expression of some members, such as TGM2, was significantly higher in tumor samples than in normal samples in pancreatic cancer (P-value significant). Figure 1 A- Figure 1 B).

[0065] 2. Correlation between TGM2 and RAS in the GEPIA database: Scatter plots showed a significant positive correlation between TGM2 expression and KRAS (R=0.59, P-value=0), NRAS (R=0.63, P-value=0), and HRAS (R=0.57, P-value=0); survival analysis showed that the overall survival of the high TGM2 expression group in lung squamous cell carcinoma, low-grade glioma, and pancreatic cancer was significantly shorter than that in the low expression group (Logrank p=0.007, 0.029, and 0.011, respectively). Figure 1 C- Figure 1 D).

[0066] 3. Prognostic effects of TGM family members on pancreatic cancer in the TCGA database: TGM1, TGM3, TGM5, and F13A1 had no statistically significant impact on overall survival (OS) and progression-free survival (PFS) in pancreatic cancer (P>0.05); TGM2 and TGM4 had significant effects on pancreatic cancer prognosis. The OS (P=0.0021) and PFS (P=0.0225) of the high-expression TGM2 group were significantly shorter than those of the low-expression group, and the OS (P=0.0038) and PFS (P<0.0001) of the high-expression TGM4 group were also significantly shorter than those of the low-expression group. Figure 1 E).

[0067] 4. Prognostic relationship between TGM2 and KRAS mutations in TCGA pancreatic cancer data: KRAS mutation (KRAS MUT Furthermore, the overall survival (OS) (P=0.0243) and progression-free survival (P=0.0134) of the TGM2 high expression group were significantly shorter than those of the KRAS group. MUT Furthermore, the TGM2 low expression group; KRAS wild-type (KRASWT At that time, the overall survival (OS) of the TGM2 high expression group showed a trend of shortening (P=0.0578), but the progression-free survival (PFS) was not significantly different (P=0.7894); comprehensive group analysis showed that KRAS MUT Furthermore, the TGM2 high expression group had the worst prognosis, KRAS WT Furthermore, the prognosis was relatively better in the TGM2 low expression group (P<0.0001). Figure 1 F).

[0068] 5. TGM2 expression in single-cell data: Cell subset analysis showed that the expression rate and average expression level of TGM2 in tumor cells were significantly higher than those in other subsets such as stellate cells and macrophages / monocytes. Figure 1 G); Spatial distribution heatmaps and violin plots show that TGM2 expression in the Mal region is significantly higher than in the Bdy and nMal regions (P<2e-16). Figure 1 H).

[0069] Example 2: TGM2 inhibitors Cystamine and GK921 inhibit pancreatic cancer cell proliferation. [1] Experimental materials 1. Cell line: Pancreatic cancer cell line Capan-1 (KRAS) G12D Mutation), Capan-2 (KRAS) G12V (mutated) and BXPC-3 cells (KRAS) WT Purchased from ATCC.

[0070] 2. Reagents: Cystamine (Sigma-Aldrich, catalog number C56-25G), GK921 (Selleck, catalog number S8166), DMEM medium (Gibco, catalog number 11965-092), fetal bovine serum (FBS, Gibco, catalog number 10099-141), CCK-8 reagent (Beyotime, catalog number C0037), anti-KRAS antibody (CST, catalog number 3965S), anti-β-Actin antibody (CST, catalog number 3700S).

[0071] [2] Experimental Procedure 1. Cell culture: Capan-1, Capan-2 and BXPC-3 cells were cultured in DMEM medium containing 10% FBS and 1% Pen-Strep at 37°C in a 5% CO2 incubator until the logarithmic growth phase.

[0072] 2. Cystamine and GK921 treatment: Cells were seeded in 96-well plates (3 × 10⁶ cells per well). 3After 24 hours, fresh culture medium containing Cystamine [0 mM (control group), 0.5 mM, 1 mM] and GK921 [0 μM (control group), 1 μM, 2 μM] was added, with 3 replicates per group, and cultured for 48 hours. Cells were then seeded into 6-well plates and low-adhesion plates (3 × 10⁶ cells per well). 3 After 24 hours, fresh culture medium containing Cystamine [0 mM (control group), 0.125 mM, 0.25 mM, 0.5 mM, 1 mM] and GK921 [0 μM (control group), 0.25 μM, 0.5 μM, 1 μM, 2 μM] was added to each group, with 3 replicates per group, and cultured for 10 days.

[0073] 3. CCK-8 assay: Add 10 μL of CCK-8 reagent to each well, incubate at 37℃ for 2 h, and measure the OD value at 450 nm using a microplate reader to calculate the proliferation inhibition rate. Plate colony assay: Aspirate the culture medium from the six-well plate, wash three times with PBS, add 1 ml of paraformaldehyde for fixation for 15 minutes, wash three times with PBS, then add 1% crystal violet for staining for 30 minutes, wash three times with PBS, and photograph under a bright-field microscope. Spheroidization assay: After 10 days of culture, photograph the cells in the low-adhesion plate directly under a bright-field microscope.

[0074] 4. Western Blot analysis: Cells treated with Cystamine [0mM (control group), 0.5mM, 1mM] and GK921 [0μM (control group), 1μM, 2μM] were collected, total protein was extracted, and KRAS protein levels were detected.

[0075] [3] Experimental Results 1. Proliferation inhibition rate: The proliferation inhibition rate, spheroidization rate, and colony formation rate of pancreatic cancer Capan-1, Capan-2, and BXPC-3 cells by Cystamine and GK921 increased with increasing concentration. Figure 2 C- Figure 2 J, Figure 2 M- Figure 2 T).

[0076] 2. KRAS protein level: In pancreatic cancer cell lines, the protein expression levels of TGM2 and KRAS are positively correlated. Figure 2 A- Figure 2 B). Compared with the control group, the KRAS protein level decreased after treatment with Cystamine and GK921, indicating that Cystamine and GK921 inhibited cell proliferation by reducing KRAS protein levels. Figure 2 K- Figure 2 L).

[0077] Example 3: TGM2 inhibitors Cystamine and GK921 inhibit pancreatic cancer cell metastasis. [1] Experimental materials 1. Cell lines: Pancreatic cancer cell lines Capan-1 (KRAS G12D mutation), Capan-2 (KRAS G12V mutation), and BXPC-3 (KRAS WT) were purchased from ATCC.

[0078] 2. Reagents: Transwell chamber (Corning, catalog number 3422), anti-Slug antibody (CST, catalog number 9585S), anti-N-CAD antibody (CST, catalog number 13116S), anti-VIME antibody (CST, catalog number 5741S), anti-ZO1 antibody (CST, catalog number 13663S).

[0079] [2] Experimental Procedure 1. Cell culture and treatment: Capan-1, Capan-2 and BXPC-3 cells were cultured in DMEM medium containing 10% FBS to the logarithmic growth phase. After serum starvation for 12 hours, serum-free medium containing Cystamine [0 mM (control group), 0.5 mM, 1 mM] and GK921 [0 μM (control group), 1 μM, 2 μM] was added and the cells were treated for 24 hours.

[0080] 2. Transwell migration assay: Resuspend the treated cells at 1×10⁶ cells / day. 5 200 μL of the culture medium containing 10% FBS was added to the upper chamber of the Transwell, and 600 μL of the medium to the lower chamber was added. The cells were cultured at 37°C for 24 h. After crystal violet staining, the number of migrating cells was counted.

[0081] 3. Western Blot analysis: Collect the processed cells, extract total protein, and detect the levels of Slug, N-CAD, VIME, and ZO1 proteins.

[0082] [3] Experimental Results 1. Migration inhibition: The migration inhibition rates of Cystamine and GK921 on Capan-1, Capan-2, and BXPC-3 cells increased with increasing concentration. Figure 3 A- Figure 3 D).

[0083] 2. EMT-related protein levels: Compared with the control group, Cystamine and GK921 treatments decreased Slug protein levels, N-CAD, and VIME levels, indicating that GK921 inhibits EMT by downregulating Slug, thereby reducing cell metastasis. Figure 3 E- Figure 3 F).

[0084] Example 4: Regulation of Ras protein expression by the TGM family and its interaction with KRAS [1] Experimental materials 1. Cell lines: 293T cells and pancreatic cancer cell line Capan-1, purchased from ATCC.

[0085] 2. Plasmids and antibodies: Flag-tagged TGM1, TGM2, TGM3, TGM4, TGM5, TGM6, and F13A1 plasmids; HA-tagged KRAS plasmids. WT KRAS G12D KRAS G12V KRAS G12R KRAS G12C KRAS G13D KRAS Q61H Plasmids; anti-Flag antibodies, anti-HA antibodies, anti-KRAS antibodies, anti-NRAS antibodies, anti-HRAS antibodies, anti-β-Actin antibodies; immunoprecipitation (IP) kit reagents.

[0086] [2] Experimental Procedure 1. Cell culture: 293T cells and Capan-1 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator until the logarithmic growth phase.

[0087] 2. Plasmid transfection: Transfect Flag-tagged TGM family plasmids (either alone or co-transfected with HA-tagged Kras plasmids) into 293T cells or Capan-1 cells, and continue culturing for 24-48 hours after transfection. For dose gradient experiments, use different concentrations (gradients) of Flag-TGM plasmids and HA-KRAS. WT The plasmid was co-transfected into 293T cells.

[0088] 3. Protein extraction and immunoprecipitation (IP): Collect transfected cells and extract total protein according to the kit instructions. During the IP experiment, use either anti-HA antibody or anti-Flag antibody to capture the protein complex, and then collect the immunoprecipitate.

[0089] 4. Western Blot detection: Total protein or IP precipitate is subjected to SDS-PAGE electrophoresis, transferred to a membrane and incubated with anti-Flag, anti-HA, anti-KRAS, anti-NRAS, anti-HRAS, and anti-β-Actin antibodies. Finally, the expression or interaction of the target protein is detected by chemiluminescence imaging.

[0090] [3] Experimental Results 1. Regulation of exogenous and endogenous Ras protein expression by the TGM family: In 293T and Capan-1 cells, transfection with Flag-TGM1, TGM2, TGM3, TGM4, TGM5, TGM6, or F13A1 plasmids showed that Western blotting revealed significantly increased levels of endogenous KRAS, NRAS, and HRAS proteins compared to the untransfected control group; when combined with HA-KRAS... WT When the dose gradient of Flag-TGM plasmid co-transfected with HA tag increased, the levels of both exogenous and endogenous KRAS proteins with the HA tag increased with increasing TGM plasmid dose. β-Actin, as an internal control protein, showed stable expression, indicating that the TGM family can increase the expression of both exogenous and endogenous Ras proteins (including KRAS, NRAS, and HRAS). Figure 4 A- Figure 4 C).

[0091] 2. The interaction between KRAS and the TGM family: Immunoprecipitation assay results showed that co-transfection with Flag-TGM1 / 2 / 3 / 4 / 5 / 6 or F13A1 and HA-KRAS... WT In the cells, co-precipitated Flag-TGM protein was detected by IP (IP:HA) with anti-HA antibody, and co-precipitated HA-Kras protein was also detected by IP with anti-Flag antibody. Normal expression of Flag-TGM, HA-KRAS, and β-Actin proteins was observed in the input sample, indicating that KRAS interacts with the entire TGM family (TGM1 to TGM6) and F13A1. Figure 4 D).

[0092] 3. Interactions between TGM1, TGM2, and different KRAS mutants: Transfection with Flag-TGM1 or Flag-TGM2 and different HA-Kras mutants (KRAS) G12D KRAS G12V KRAS G12R KRAS G12C KRAS G13D KRAS Q61H Following this, Western blot analysis showed successful expression of Flag-TGM1 and TGM2 proteins, and that all HA-Kras mutants interacted with TGM1 and TGM2. Simultaneously, the levels of related KRAS proteins changed in the presence of TGM, while β-Actin protein expression remained stable, suggesting that TGM1 and TGM2 interact with various KRAS mutants and affect their expression. Figure 4 E- Figure 4 F).

[0093] Example 5: TGM2 regulates KRAS ubiquitination degradation via a non-classical enzymatic pathway [1] Experimental materials Cell lines: 293T cells and pancreatic cancer cell line Capan-1, purchased from ATCC.

[0094] Plasmids and reagents: Flag-TGM2 plasmid, HA-KRAS WT Plasmids, His-Ub plasmid; TGM inhibitors Cystamine and GK921; proteasome inhibitor MG132; immunoprecipitation (IP) kit reagents; anti-HA antibody, anti-Flag antibody, anti-Ub antibody, anti-β-Actin antibody; Flag-TGM1 with a mutation at a classic transaminase functional site. C375S Flag-TGM2 C277S Flag-TGM3 C273S Flag-TGM4 C285S Flag-TGM5 C285S Flag-TGM6 C274S Flag-F13A1^C315S plasmid, Flag-EPB4.2 plasmid.

[0095] [2] Experimental Procedure 1. Cell culture: 293T cells and Capan-1 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator until the logarithmic growth phase.

[0096] 2. Plasmid transfection and drug treatment: Ubiquitination experiment: Flag-TGM2 (dose gradient), HA-KRAS WT His-Ub plasmid was co-transfected into 293T or Capan-1 cells, and MG132 was added for treatment at the same time; in some experiments, TGM inhibitor Cystamine or GK921 was added after transfection.

[0097] Functional site mutation experiment: TGM family plasmids with mutated functional sites of classical transaminases (such as Flag-TGM1) were used. C375S (etc.) and HA-KRAS WT The plasmids were co-transfected into 293T or Capan-1 cells, and wild-type TGM plasmids (Flag-TGM1, etc.) and negative control (NC) groups were set up; Flag-EPB4.2 and HA-KRAS were also transfected. WT Co-transfection was used as a control.

[0098] Immunoprecipitation (IP) and Western Blot detection: After collecting cells, total protein was extracted. Protein complexes were captured by IP experiment using anti-HA or anti-Flag antibodies. Then, Western Blot was used to detect the expression or interaction of proteins such as Ub (ubiquitination level), HA (Kras), and Flag (TGM family), with β-Actin as an internal control.

[0099] [3] Experimental Results 1. Regulation of KRAS ubiquitination by TGM2: In 293T and Capan-1 cells, co-transfection with Flag-TGM2 (dose gradient) and HA-KRAS... WT After adding His-Ub and MG132, anti-HA antibody IP assay showed that the ubiquitination (Ub) level of KRAS decreased with increasing Flag-TGM2 dose; the protein levels of HA-Kras and Flag-TGM2 in the input changed with TGM2 dose, while β-Actin expression remained stable, suggesting that TGM2 can reduce the ubiquitination level of KRAS. Figure 5 A- Figure 5 B).

[0100] 2. Effects of TGM inhibitors on KRAS ubiquitination and TGM2-KRAS interaction: The addition of TGM inhibitors Cystamine or GK921 compared to transfections only with Flag-TGM2 and HA-KRAS... WT Compared to the control group, the HA-Kras protein level detected by anti-Flag antibody IP was decreased; in the ubiquitination assay (anti-HA antibody IP), the KRAS ubiquitination level recovered; the expression of all proteins in the input was normal, indicating that Cystamine and GK921 can affect the interaction between TGM2 and KRAS and regulate KRAS ubiquitination. Figure 5 C- Figure 5 D).

[0101] 3. The interaction between mutations at the functional sites of classic TGM transaminases and KRAS: In 293T and Capan-1 cells, transfection with TGM family plasmids (such as TGM1) carrying mutations at classical transaminase functional sites was performed. C375S TGM2 C277S (etc.) and HA-KRAS WT Subsequently, Western blot analysis showed that the Flag tag confirmed normal expression of the mutant protein, and the interaction between KRAS and mutant TGM was not significantly inhibited (HA signaling was not significantly weakened compared to the wild-type TGM group); no related signal was found in the negative control (NC) group, and Flag-EPB4.2 and HA-KRAS were also observed. WTThe interaction pattern differs from that of the TGM family, indicating that mutations at the classical transaminase functional sites of TGM cannot inhibit its interaction with KRAS. Figure 5 E- Figure 5 F).

[0102] Comparative Example 1: Effects of Classical and Non-Classical TGM2 Enzyme Inhibitors on KRAS Ubiquitination [1] Experimental Objective Compare the inhibitory effects of TGM2 classical transglutaminase activity inhibitors (such as Cystamine) and non-classical enzyme activity inhibitors (such as GK921) on KRAS ubiquitination to verify whether TGM2 regulation of KRAS ubiquitination depends on classical enzyme activity.

[0103] [2] Experimental materials and procedures 1. Cell line: 293T cells, transfected with HA-KRAS WT+His-Ub plasmid.

[0104] 2. Inhibitors: Cystamine (classic enzyme activity inhibitor, 0.5mM), GK921 (non-classic enzyme activity inhibitor, 1μM), MDC (another classic enzyme activity inhibitor, 10μM).

[0105] 3. Experimental groups: ① Control group; ② Cystamine group; ③ GK921 group; ④ MDC group; After 48 h of treatment, MG132 (10 μM) was added and treated for 6 h. Cells were collected and KRAS ubiquitination level was detected by Co-IP.

[0106] [3] Experimental Results The control group's KRAS ubiquitination level was set at 100%. The Cystamine group showed a decrease of 58.3%±4.7%, the MDC group a decrease of 52.1%±4.3%, and the GK921 group a decrease of 62.5%±5.1%. All three groups showed significant differences compared to the control group (p<0.01), but there were no significant differences among the three groups (p>0.05). This indicates that both classical and non-classical enzyme activity inhibitors can inhibit the regulatory effect of TGM2 on KRAS ubiquitination, further confirming that TGM2 regulates KRAS ubiquitination through the non-classical enzyme activity pathway (classical enzyme activity inhibitors may indirectly affect the TGM2 conformation rather than directly inhibit classical enzyme activity, thereby blocking its non-classical function).

[0107] Comparative Example 2: Effects of TGM2 and other TGM family members on KRAS ubiquitination [1] Experimental Objective The effects of different members of the TGM family (TGM1, TGM2, TGM3, TGM4, TGM5, TGM6, F13A1) on KRAS ubiquitination were compared to verify the role of TGM2.

[0108] [2] Experimental materials and procedures 1. Cell line: 293T cells, transfected with HA-KRAS WT+His-Ub plasmid, and simultaneously transfected with Flag-TGM1, Flag-TGM2, Flag-TGM3, Flag-TGM4, Flag-TGM5, Flag-TGM6, and Flag-F13A1 plasmids (the control group was transfected with an empty Flag plasmid).

[0109] 2. Experimental procedure: MG132 was added for 6 hours after transfection 48 hours later. Cells were collected, KRAS ubiquitination level was detected by Co-IP, and the expression of each Flag-TGM protein was detected by Western Blot.

[0110] [3] Experimental Results Compared with the control group, the KRAS ubiquitination level decreased by 62.3% ± 5.2% in the Flag-TGM2 transfection group (p < 0.01); the KRAS ubiquitination levels in the Flag-TGM1, Flag-TGM3, Flag-TGM4, Flag-TGM5, Flag-TGM6, and Flag-F13A1 transfection groups all decreased to varying degrees. This indicates that the entire TGM family can inhibit KRAS ubiquitination degradation, providing a theoretical basis for targeted TGM family therapy in KRAS-related cancers.

[0111] Based on their different research directions, relevant staff members may consider the following alternative solutions: 1. TGM2 siRNA / shRNA interference technology: This technique specifically downregulates TGM2 expression using small interfering RNA (siRNA) or short hairpin RNA (shRNA), replacing TGM2 inhibitors to reduce KRAS protein levels and inhibit tumor progression. The advantages of this approach are high specificity and avoidance of off-target effects from inhibitors; the disadvantages are the need for transfection via viral vectors or liposomes, resulting in low delivery efficiency and limiting clinical application (e.g., difficulty penetrating solid tumors).

[0112] 2. TGM2 Antibody-Drug Conjugates (ADCs): These conjugate TGM2-specific antibodies with cytotoxic drugs (such as microtubule inhibitors). The antibodies target and bind to tumor cells that highly express TGM2, releasing the toxic drug to kill the tumor cells. The advantages of this approach are strong targeting and low cytotoxicity to normal cells; the disadvantages are high preparation cost, susceptibility to antibody resistance, and the inability to directly regulate KRAS ubiquitination, making it only suitable for tumors with high TGM2 expression.

[0113] 3. KRAS ubiquitination promoters: By screening for small molecule compounds that can activate E3 ubiquitin ligases (such as WWP1) or inhibit deubiquitinating enzymes (such as USP7), KRAS ubiquitination degradation can be promoted, replacing TGM2 inhibitors. The advantage of this approach is that it can directly act on the KRAS ubiquitination process; the disadvantages are that some E3 ubiquitin ligases / deubiquitinating enzymes also play important physiological functions in normal cells, targeted regulation is prone to side effects, and the applicability is limited by the type of KRAS mutation.

[0114] 4. TGM2 and KRAS Interaction Inhibitors: Small molecule compounds that can block the interaction between TGM2 and KRAS are screened using computer simulations to replace TGM2 enzyme activity inhibitors and inhibit the regulatory effect of TGM2 on KRAS ubiquitination. The advantage of this approach is that it acts directly on the interaction interface, resulting in higher specificity; the disadvantages are that the binding structure of TGM2 and KRAS needs to be clearly defined, making screening difficult, and the compounds may have weak cell membrane penetration ability, affecting therapeutic efficacy.

[0115] While the aforementioned alternatives can achieve similar objectives, they all have their own limitations (such as low delivery efficiency, high cost, and significant side effects). In contrast, the TGM2 inhibitor regimen of this invention has advantages such as wide applicability (covering multiple KRAS mutation types), high safety (targeting tumor cells with high TGM2 expression), and comprehensive efficacy (simultaneously inhibiting proliferation and metastasis), making it more valuable for clinical application.

[0116] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of TGM2 inhibitors in the preparation of drugs that regulate KRAS ubiquitination.

2. The application according to claim 1, characterized in that, The regulation is positive, which increases the level of KRAS ubiquitination.

3. The application according to claim 2, characterized in that, The TGM2 inhibitors include Cystamine and GK921.

4. The application according to claim 3, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers.

5. Application of TGM2 inhibitors in the preparation of drugs for the prevention and treatment of KRAS-related cancers.

6. The application according to claim 5, characterized in that, KRAS gene mutations are frequently expressed in the aforementioned KRAS-related cancers.

7. The application according to claim 6, characterized in that, The TGM2 inhibitor prevents and treats KRAS-related cancers by increasing KRAS ubiquitination levels.

8. The application according to claim 7, characterized in that, The TGM2 inhibitors include Cystamine and GK921.

9. The application according to claim 8, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers.

10. Application of TGM2 detection reagent in the preparation of KRAS-related cancer prognostic assessment products.

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