Method for screening KRAS mutant interacting protein

Abstract

The invention relates to the field of biomedicine, and discloses a method for screening KRAS mutant interacting proteins, an HEK293T cell line for continuously expressing an HA-Turbo ID-KRAS variant is established by utilizing lentivirus transduction, the stability of transgene expression is ensured through puromycin screening, and variation caused by transient transfection is reduced as much as possible. After a Turbo ID mediated proximity marker is activated by biotin treatment, KRAS proximity binding proteins are covalently biotinylated, captured by NeutrAvidin bead purification, and analyzed by quantitative mass spectrometry. According to the invention, two potential KRAS mutation treatment target proteins of LZTR1 and LAMTOR1 can simultaneously inhibit activation and efficacy exertion of three different mutants of G12C, G12D and G12V of the G12 mutant, compared with drugs on the market that a single target of G12C directly targets the KRAS mutant, a brand new KRAS mutation targeting intervention strategy is provided, the clinical curative effect can be further improved, and the occurrence rate of secondary drug resistance can be effectively reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for screening interaction proteins of KRAS mutants. Background Art

[0002] As the earliest discovered proto-oncogene in humans, RAS exhibits tissue distribution mutations in approximately 20% of malignant tumors, including pancreatic cancer, colon cancer, and non-small cell lung cancer. Among them, KRAS constitutes the main mutant subtype, accounting for approximately 75% of RAS-driven tumorigenesis. Among KRAS mutations, the mutation at codon 12 dominates in clinical cases, and G12D (36%), G12V (23%), and G12C (14%) together account for more than 60% of KRAS variations. These mutations not only serve as biomarkers for disease staging but also significantly affect the overall survival rate (OS) of patients during clinical cancer treatment. Due to its special molecular characteristics, the therapeutic targeting of KRAS remains challenging: a compact globular structure composed of only 189 amino acids (molecular weight approximately 21 kDa); the protein surface is smooth, lacking deep hydrophobic pockets for small molecule binding. In addition, KRAS has a strong picomolar affinity for GTP / GDP. When KRAS binds to GDP, it is in an inactivated state, and when it binds to GTP, it turns into an activated state, activating the downstream signaling pathway. Conventionally, nucleotides are abundant in cells, and KRAS has a natural competitive advantage in binding to GTP / GDP. In summary, KRAS was once considered an "undruggable" target in the field of tumor drug discovery.

[0003] In the past three years, certain breakthroughs have been made in the drug research and development of KRAS G12C. However, to date, only four drugs, namely Sotorasib, Adagrasib, Fulzerasib, and Garsorasib, have been approved for marketing at home and abroad, which still cannot meet the huge clinical needs. There is an urgent need to develop new druggable target strategies for KRAS G12 mutations.

[0004] Currently, there are 4 approved inhibitors targeting the KRAS G12C subtype: 1) They all target G12C (which only accounts for 14% of G12 mutations), cannot target G12D and V subtypes, and covalently bind to the cysteine of the KRAS G12C mutation, locking the KRAS G12C mutation in an inactivated state bound to GDP, thereby preventing downstream signal pathway transduction. 2) They only target the G12C subtype and are prone to secondary drug resistance. (The drug resistance mechanism of KRAS G12C inhibitors can be referred to the article "Diverse alterations associated with resistance to KRAS(G12C)inhibition" in Nature). One of the important reasons for drug resistance is the existence of cell clones with other mutant subtypes of G12.

[0005] Due to the compact globular structure of KRAS (with a molecular weight of about 21 kDa), the protein surface is smooth and lacks deep hydrophobic pockets for small molecule binding, so it is still very difficult to directly develop targets against the KRAS mutant itself. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies of the prior art and provide a method for screening proteins that interact with KRAS mutants.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for screening proteins that interact with KRAS mutants. Using lentiviral transduction, we established HEK293T cell lines that continuously express HA-TurboID-KRAS variants (WT, G12C / D / V), and ensured the stability of transgenic expression through puromycin screening, while minimizing variations caused by transient transfection. The experimental design includes four groups of KRAS variants (HA-TurboID-KRAS fusion proteins) and a control group (only HA-TurboID). After biotin treatment to activate TurboID-mediated proximity labeling, KRAS proximal proteins are covalently biotinylated, captured by purification with NeutrAvidin beads, and analyzed by quantitative mass spectrometry. This method successfully resolved the different interactomes between KRAS-WT and G12 mutants, and simultaneously revealed their specific metabolic pathway alterations.

[0009] Specifically, the construction of KRAS wild-type and G12 C / D / V mutant stable cell lines includes plasmid construction and sequence verification, plasmid transformation and amplification, lentiviral infection and biological activity verification.

[0010] Specifically, the TurboID proximity labeling technique is used to enrich proteins that interact with KRAS.

[0011] Specifically, the quantitative proteomic characteristics of KRAS interacting proteins include LC-MS / MS proteomic analysis, data processing, and bioinformatics analysis: The raw mass spectrometry data was analyzed by ProteinPilot software, and the experimental MS / MS secondary spectra were matched with the theoretical spectra through the NCBI database to initially obtain reliable peptide information. Subsequently, the ProteinProspector platform and SwissProt database were used to perform a secondary search on the peptide information library. These two-stage analyses yielded basic protein characteristics, including protein accession numbers, gene names, unique identifiers, peptide counts, and protein names. Through Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, functional annotation of the interaction networks of KRAS wild-type and mutants was performed. Meanwhile, the STRING was used to construct a protein-protein interaction network (PPI). Finally, through differential expression analysis with the TCGA database, the expression patterns of target proteins in the context of malignant tumors were verified.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. The present invention ingeniously employs the TurboID proximity labeling technology in combination with proteomic techniques. The experimental results show that multiple intracellular metabolic pathways respond to the KRAS G12 mutation, and different metabolic remodeling is observed under different mutation types. And a very important finding is that the three G12 mutants, G12C, G12D, and G12V, have some common interacting proteins, which play an important restrictive role in the functional activation and exertion of KRAS G12C;

[0014] 2. The two key regulatory proteins discovered by the present invention are: LZTR1 and LAMTOR1 proteins. These two proteins can simultaneously affect the activation functions of the three different mutants, G12C, G12D, and G12V, of the G12 mutant, and can be used as potential druggable targeting strategies. Compared with the currently marketed G12C-targeted drugs, they have the characteristics of a wider acting target and stronger potential effects;

[0015] 3. The LZTR1 and LAMTOR1 proteins can simultaneously inhibit the activation of the three different mutants, G12C, G12D, and G12V, of the G12 mutant. Compared with the single G12C target of the marketed drugs, it can effectively reduce secondary drug resistance.

[0016] 4. The druggability strategy targeting LZTR1 and LAMTOR1 proteins is relatively simple. Compared with directly targeting KRAS mutants (whose three-dimensional structures determine their difficulty in being drugged), it can reduce the time and difficulty of drug development.

[0017] 5. The TurboID proximity labeling technology combined with proteomics technology is universal. Among KRAS G12 mutations, G12D (36%), G12V (23%) and G12C (14%) account for more than 60%. There are still other rare mutations, such as G12V, etc. A new targeted strategy for drug development can be carried out according to our method. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a flowchart of the present invention.

[0019] Figure 2 It is a schematic diagram of the key experimental results of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Please refer to Figure 1-2 , the present invention provides a technical solution:

[0022] A method for screening KRAS mutant interacting proteins, characterized by comprising the following steps:

[0023] Construct a stable cell line expressing HA-TurboID-KRAS fusion protein, wherein the KRAS is selected from wild-type WT and G12C, G12D, G12V mutant types;

[0024] Activate TurboID-mediated proximity labeling with biotin to covalently biotinylate KRAS neighboring proteins;

[0025] Purify biotinylated proteins through NeutrAvidin beads and perform LC-MS / MS quantitative proteomics analysis;

[0026] Screen for differential proteins that specifically bind to KRAS mutants through bioinformatics.

[0027] The construction of the stable cell line includes: transfecting the HA-TurboID-KRAS expression vector into HEK293T cells by lentivirus, and screening with puromycin to obtain a cell line stably expressing the fusion protein.

[0028] The bioinformatics analysis includes: constructing a protein-protein interaction network through the STRING database, performing KEGG pathway analysis and GO function annotation, and verifying the differential expression of target proteins in malignant tumors by combining with the TCGA database.

[0029] The differential proteins include: the LZTR1 protein with weakened binding to the KRAS mutant, and the LAMTOR1 protein with enhanced binding to the KRAS mutant.

[0030] Restore the regulatory target of KRAS ubiquitination degradation by enhancing the activity of the LZTR1 protein or its coding gene; block the regulatory target of the mTORC1 signaling pathway by inhibiting the activity of the LAMTOR1 protein or its coding gene.

[0031] The KRAS mutation includes at least one of G12C, G12D, and G12V.

[0032] An agonist capable of enhancing the binding activity of the LZTR1 protein to the KRAS mutant, which inhibits tumor proliferation by restoring the KRAS ubiquitination degradation pathway; an inhibitor capable of blocking the binding activity of the LAMTOR1 protein to the KRAS mutant, which reverses metabolic reprogramming and immune escape by inhibiting the mTORC1 signaling pathway.

[0033] The drug exerts its effect by regulating the insulin signaling pathway, the reactive oxygen species metabolism pathway, or the glycolipid metabolism pathway.

[0034] KRAS mutation detection module, target protein expression level detection module (detecting LZTR1 and LAMTOR1), drug selection module (selecting LZTR1 agonist and / or LAMTOR1 inhibitor according to the detection results).

[0035] The detection module includes: a TurboID proximity labeling technology unit, a quantitative proteomics analysis unit, and a KRAS mutation-specific interaction network analysis unit.

[0036] Example 1, Construction of KRAS wild-type and G12 C / D / V mutant stable cell lines:

[0037] 1. Plasmid construction and sequence verification

[0038] We designed an affinity-purified HA tag tandem TurboID at the N-terminus of KRAS through PCR homologous recombination. Using the BamHI (Sangon Biotech, Shanghai) single restriction enzyme cloning strategy (the restriction site was selected according to the conserved domain of KRAS in the GenBank record), the recombinant fragment was cloned into the pLVX lentiviral vector. All plasmid constructs were sequenced by Sanger (Genewiz, Suzhou), and then the sequences were aligned with the NCBI KRAS reference sequence (NM_004985.4) using SnapGene v6.0.2. The verified HA-TurboID-KRAS WT plasmid was transformed into Stbl3 chemically competent cells (TransGen Biotech, Beijing) for large-scale propagation. Primer design involved four specific sequences (Genewiz, Suzhou):

[0039]

[0040] To generate the G12 mutants, we used the QuickMutation TM Kit (D0206S, Beyotime Biotechnology) and eight custom primers to perform site-directed mutagenesis on the wild-type (WT) plasmid:

[0041] Subtype Mutation Base Sequence (5′-3')

[0042]

[0043] By introducing nucleotide substitutions, mutations of glycine to valine / cysteine / aspartic acid were generated at codon 12, corresponding to the G12C (GGT→TGT), G12D (GGT→GAT), and G12V (GGT→GTT) variants, respectively.

[0044] After plasmid construction was completed, heat shock transformation was performed using Stbl3 competent cells. The transformed colonies were cultured in LB (Luria-Bertani) liquid medium containing ampicillin (working concentration 1:1000) under aerobic conditions (37°C) for 14 - 16 hours to achieve high-copy number plasmid amplification. The cell pellets were collected after centrifugation (4000 rpm, 10 minutes, 4°C), and then the plasmids were extracted using the HiPure Plasmid Miniprep Kit (DC201-01, Vazyme Biotechnology). The purified plasmids were eluted with 50 μL of elution buffer and stored frozen at -20°C for later use.

[0045] 2. Lentiviral Infection and Bioactivity Verification

[0046] HEK293T cells were used for lentivirus production. The cells were seeded in 10-cm culture dishes and cultured in fresh Dulbecco's Modified Eagle Medium (DMEM, 319-075-CL, Wisent), supplemented with 10% fetal bovine serum (FBS, FSD500, ExCell) and 1% streptomycin / penicillin (C100C5, CELLSAVING). For lentivirus packaging, a plasmid mixture containing the KRAS expression vector (or a plasmid containing only the tag as a blank control), psPAX2, and pMD2.0G was prepared at a mass ratio of 3:2:1, and three volumes of polyethyleneimine (PEI, Solarbio) was used as a co-transfection reagent. The viral supernatant was collected 48 hours after transfection and filtered through a 0.45-μm filter membrane. For infection, HEK293T cells seeded in 6-well plates were treated with the viral supernatant containing 8 μg / mL Polybrene as a co-infection reagent. After two rounds of infection, the cells were treated with 2 μg / mL puromycin for 72 hours to screen for stable cell lines. The surviving cells expressing HA-TurboID (tag only, blank control) and HA-TurboID-KRAS (WT, G12C, G12D, G12V) were expanded for subsequent experiments. Western blot analysis of whole cell lysates confirmed the stable overexpression of HA-TurboID and HA-TurboID-KRAS (WT, G12C, G12D, G12V). The antibodies used were HA (AE105, Abclonal) and β-actin (AC026, Abclonal). To verify the biological activity, a Cell Counting Kit-8 (CCK-8, CK04, Dojindo) assay was performed on five experimental groups: blank control, WT, G12C, G12D, G12V. Each group included six technical replicates.

[0047] Example 2. Enrichment of KRAS interacting proteins using TurboID proximity labeling technology

[0048] The stable cell lines obtained from the previous experiment were expanded to a density of 1.6×10 8 cells. 50 μM biotin was added to the medium and incubated for 2 hours (the blank group was incubated for only 0.5 hours), and the KRAS proximal proteins were biotinylated by the activity of the TurboID biotin ligase. NeutrAvidin TM agarose resin (Thermo Scientific, Cat#31000) was used to affinity purify the biotinylated proteins by rotating at 4°C for 14 hours.

[0049] Subsequently, whole cell lysates (input) and NeutrAvidin-enriched proteins (AP) were analyzed by Western blot. The antibodies used were HA and HRP-conjugated streptavidin (SA00001-0, Proteintech).

[0050] Example 3, Quantitative Proteomic Characterization of KRAS-Interacting Proteins

[0051] 1. Sample Preparation

[0052] The enriched proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PG112, Epizyme) and visualized with Coomassie Brilliant Blue R-250. Each protein band was cut into 1 mm 3 gel fragments for subsequent mass spectrometry sample pretreatment. Decolorization was performed with 25 mM ammonium bicarbonate containing 5% acetonitrile (ACN). Disulfide bonds were reduced with 10 mM dithiothreitol (DTT) at 56 °C for 45 minutes, followed by alkylation with 20 mM iodoacetamide (IAM) in the dark for 45 minutes.

[0053] Trypsin digestion was carried out at 37 °C for 14 - 16 hours at a ratio of 1:100 in 25 mM ammonium bicarbonate (NH4HCO3). The digestion was terminated with 10% formic acid (FA), and the peptides were extracted with 50% acetonitrile (ACN) / 5% formic acid. The peptide solution was concentrated to dryness using a CentriVap benchtop centrifugal concentrator and reconstituted with 3% acetonitrile (ACN) / 2% formic acid prior to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0054] 2. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) Analysis

[0055] Samples were analyzed using an ACQUITY UPLC M-Class system (Waters) coupled with a ZenoTOF 7600 mass spectrometer (SCIEX). The mass spectrometer used electron activation dissociation (EAD) and Zeno TM Trap technology to improve sensitivity. The chromatographic separation buffers were 0.1% formic acid (FA) / 2% acetonitrile (ACN) aqueous solution (buffer A) and 0.1% formic acid / 2% acetonitrile aqueous solution (buffer B). The mobile phase was linearly gradient separated at a flow rate of 5 μL / min. The peptide resolution was loaded into a chromatographic injection vial (VDAP-4025PBS-631-100, CNW, China), and the sample injection volume was 4 μl each time. The mass spectrometry data was acquired in data-dependent acquisition (DDA) mode. The reagents used for sample pretreatment and loading were all of mass spectrometry grade and purchased from Sigma-Aldrich (MO).

[0056] 3. Data processing and bioinformatics analysis

[0057] The raw mass spectrometry data was analyzed using ProteinPilot software, and the experimental MS / MS spectra were matched with the theoretical spectra through the NCBI database to initially obtain reliable peptide information. Subsequently, the ProteinProspector platform was used to perform a secondary search of the peptide information database in combination with the SwissProt database (version number 2021.06.18; Homo sapiens). These two-stage analyses yielded basic protein characteristics, including protein accession numbers, gene names, unique identifiers, peptide counts, and protein names.

[0058] Functional annotations of the interaction partners of KRAS wild-type and mutants were performed through Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Meanwhile, a protein-protein interaction network (PPI) was constructed using STRING (string-db.org / ). Bubble plots were drawn using the R package ggplot2 (v3.5.1), and set analysis was performed using Venn Diagram (v1.7.3) to achieve data visualization. Finally, the expression pattern of the target protein in the context of malignant tumors was verified through differential expression analysis with the TCGA database (https: / / cancergenome.nih.gov / ).

[0059] It should be noted that the technical problem to be solved by the technical solution of the present invention is that carcinogenic RAS mutations continuously maintain the GTP-bound activation state, leading to downstream signal transduction disorders, ultimately disrupting cellular homeostasis and inducing cellular malignant transformation. The present invention combines the TurboID proximity labeling technique with quantitative proteomics LC-MS / MS to systematically characterize the proximity-binding proteins of wild-type KRAS and three high-frequency carcinogenic mutant subtypes G12C, G12D, and G12V. Through comprehensive bioinformatics analysis of the mutant-specific interaction network and different metabolic pathways, we found that mutant KRAS-binding proteins were significantly enriched in aspects such as the insulin signaling pathway, reactive oxygen species-related pathways, glucose and lipid metabolism. The metabolic reprogramming pathways of KRAS G12 mutations jointly promoted tumor proliferation and immune escape. Subsequently, by analyzing the similarity of the proximity-binding proteins in the three G12 C / D / V mutants, we identified two specific proteins: 1) the LZTR1 protein (KRAS E3 ubiquitin ligase adapter), which showed a significantly weakened binding to KRAS mutants after RAS mutation. 2) The LAMTOR1 protein (an important regulator of mTORC1), which showed a significantly enhanced binding to KRAS G12 mutants after RAS mutation. This multi-dimensional analysis depicted a comprehensive map of the interaction network of KRAS WT and G12 mutants, revealing the metabolic reprogramming pathways associated with KRAS activating mutations. The results of our analysis provide potential therapeutic targets for KRAS-driven tumorigenesis and establish a mechanistic framework for developing specific therapeutic strategies against KRAS mutations. The LZTR1 protein and the LAMTOR1 protein, these two proteins, can be candidate intervention targets for KRAS mutation-related tumor treatment. By targeting these two proteins, the functional activation of KRAS mutations can be reversed, thereby producing tumor treatment efficacy.

[0060] Core step: Construct a HEK293T cell line that continuously expresses HA-TurboID-KRAS variants (WT, G12C / D / V). After biotin treatment to activate TurboID-mediated proximity labeling, KRAS proximity proteins are covalently biotinylated, captured by purification with NeutrAvidin beads, and analyzed by quantitative mass spectrometry.

[0061] This specific embodiment is only an interpretation of the present invention and not a limitation thereof. Those skilled in the art can make modifications to this embodiment without creative contributions according to their needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for screening KRAS mutant interacting proteins, characterized in that, Comprising the following steps: Construct a stable cell line expressing the HA-TurboID-KRAS fusion protein, wherein the KRAS is selected from wild-type WT and G12C, G12D, G12V mutant types; Activate TurboID-mediated proximity labeling with biotin to covalently biotinylate KRAS-proximal proteins; Purify the biotinylated protein through NeutrAvidin beads and perform LC-MS / MS quantitative proteomics analysis; Screen for differential proteins that specifically bind to KRAS mutants through bioinformatics.

2. The method for screening KRAS mutant interacting proteins according to claim 1, wherein The construction of the stable cell line includes: transfecting HEK293T cells with the HA-TurboID-KRAS expression vector by lentivirus and screening with puromycin to obtain a cell line stably expressing the fusion protein.

3. A method for screening KRAS mutant interacting proteins according to claim 1, characterized in that, The bioinformatics analysis includes: constructing a protein interaction network through the STRING database, performing KEGG pathway analysis and GO function annotation, and verifying the differential expression of target proteins in malignant tumors by combining with the TCGA database.

4. The method for screening KRAS mutant interacting proteins according to claim 1, characterized in that, The differential proteins include: the LZTR1 protein with weakened binding to the KRAS mutant, and the LAMTOR1 protein with enhanced binding to the KRAS mutant.

5. A drug target for treating KRAS mutation-related tumors, characterized in that, The targets are: regulatory targets for restoring KRAS ubiquitination and degradation by enhancing the activity of the LZTR1 protein or its coding gene; regulatory targets for blocking the mTORC1 signaling pathway by inhibiting the activity of the LAMTOR1 protein or its coding gene.

6. The drug target for treating KRAS mutation-related tumors according to claim 5, wherein: The KRAS mutation includes at least one of G12C, G12D, G12V.

7. A pharmaceutical composition for treating KRAS mutation-related tumors, characterized in that: An agonist capable of enhancing the binding activity of the LZTR1 protein to the KRAS mutant, which inhibits tumor proliferation by restoring the KRAS ubiquitination and degradation pathway; an inhibitor capable of blocking the binding activity of the LAMTOR1 protein to the KRAS mutant, which reverses metabolic reprogramming and immune escape by inhibiting the mTORC1 signaling pathway.

8. A pharmaceutical composition for treating KRAS mutation-related tumors according to claim 7, wherein: The drug exerts its effect by regulating the insulin signaling pathway, the reactive oxygen metabolism pathway, or the glycolipid metabolism pathway.

9. A treatment system for KRAS mutation-related tumors, characterized in that: KRAS mutation detection module, target protein expression level detection module (detecting LZTR1 and LAMTOR1), drug selection module (selecting LZTR1 agonist and / or LAMTOR1 inhibitor according to the detection results).

10. The treatment system for KRAS mutation-related tumors according to claim 9, wherein The detection module includes: a TurboID proximity labeling technology unit, a quantitative proteomics analysis unit, and a KRAS mutation-specific interaction network analysis unit.

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