Computer-aided drug screening method, system and device based on PTK6 / hnRNPH1 / NBR1

Abstract

The invention discloses a computer-aided drug screening method, system and device based on PTK6 / hnRNPH1 / NBR1, relates to the field of bioinformatics and biomedicine, provides a computer-aided drug screening method based on PTK6 and hnRNPH1, and provides a computer-aided drug screening system and device based on PTK6 and hnRNPH1 and a computer readable storage medium. The invention also provides application of the PTK6 / hnRNPH1 in treating the colorectal cancer and screening medicines related to the colorectal cancer, a pharmaceutical composition and a method.

Description

Technical Field

[0001] The present application relates to the fields of bioinformatics and biomedicine, and specifically to a computer-assisted drug screening method, system and device based on PTK6 / hnRNPH1 / NBR1. Background Art

[0002] In the past two decades, a large number of studies have shown that the activation of autophagy may promote the malignant progression of cancer. Under normal circumstances, autophagy can recycle damaged components to maintain the homeostasis of the intracellular environment. However, during tumor progression, autophagy can provide nutrients to tumor cells, thereby promoting migration, invasion, or adapting to nutrient deficiency and hypoxic environments. At the same time, more and more studies are focusing on the significance of autophagy in colorectal cancer (CRC) and its potential as a target for therapeutic intervention. Although chloroquine (CQ) and other autophagy-targeting drugs have shown the effect of inhibiting autophagic lysosomal degradation in CRC, they have not yet been approved for clinical use. Therefore, further exploring the specific molecular mechanisms of autophagy in CRC plays an important role in developing new treatment strategies and improving patient prognosis.

[0003] Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase with SH3, SH2 domains and tyrosine kinase catalytic (TyrKc) domains, and participates in signal transduction by phosphorylating a variety of substrate proteins. PTK6 is involved in regulating the proliferation, apoptosis and migration of a variety of tumor cells, but its role and molecular mechanism in the autophagy process of CRC remain unclear.

[0004] Phase separation refers to the dynamic liquid-liquid phase separation (LLPS) structure formed by the interaction of specific proteins or nucleic acid molecules, which plays a vital role in cell signaling and gene expression regulation. LLPS depends on intrinsically disordered regions (IDRs) inside proteins, which drive the formation of membraneless organelles through multivalent weak interactions. However, the specific function and mechanism of LLPS in CRC autophagy still need to be further explored. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0006] The present invention provides a computer-aided drug screening method based on PTK6 and hnRNPH1, the method comprising: obtaining PTK6 protein and hnRNPH1 protein data; selecting the spatial structure of the PTK6 protein and hnRNPH1 protein complex, and determining the complex binding site as the binding site of the targeted drug;

[0007] A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

[0008] Furthermore, the computer-assisted drug screening method comprises the following specific steps:

[0009] Obtain the binding site of PTK6 protein and hnRNPH1 protein complex;

[0010] Based on the spatial structure of the binding site of the PTK6 protein and hnRNPH1 protein complex, compounds with similar structures were screened in the molecular database;

[0011] Molecular docking of the screened compounds with the hnRNPH1 protein was performed to calculate the affinity / binding energy of the target receptor to obtain a score, and candidate drugs were obtained by sorting according to the score.

[0012] Furthermore, the binding site of the PTK6 protein and hnRNPH1 protein complex is the 210th amino acid of the hnRNPH1 protein.

[0013] Furthermore, the computer-assisted drug screening method screens protein analogs / antibodies / RNA drugs based on the active sites of the PTK6 protein and hnRNPH1 protein complex; or screens protein analogs / antibodies / RNA drugs similar to the PTK6 protein to obtain candidate drugs.

[0014] Furthermore, the active sites include phosphorylation sites, acetylation sites, and methylation sites.

[0015] Furthermore, the computer-assisted drug screening method comprises the following specific steps:

[0016] Obtain the binding site of PTK6 protein and hnRNPH1 protein complex;

[0017] Based on the spatial structure of the binding site of the PTK6 protein and hnRNPH1 protein complex, compounds with similar structures were screened in the molecular database;

[0018] Molecular docking of the screened compounds with the PTK6 protein was performed to calculate the affinity / binding energy of the target receptor to obtain a score, and candidate drugs were obtained by ranking according to the score.

[0019] Furthermore, the computer-assisted drug screening method screens protein analogs / antibodies / RNA drugs based on the active sites of the PTK6 protein and hnRNPH1 protein complex; or screens protein analogs / antibodies / RNA drugs similar to the hnRNPH1 protein to obtain candidate drugs.

[0020] In some embodiments, the molecular databases used in virtual screening are mainly the following: ZINC: contains more than 250 million commercially available compounds for small molecule virtual screening. PubChem: contains biologically active substances for small molecule virtual screening. DrugBank: contains drugs and small molecules for drug design and discovery. ChEMBL: contains small molecules for drug discovery and chemical genomics research. ChemDB: contains a large number of known small molecules for chemical genomics research and drug discovery. HMDB: contains a large number of known small molecules for chemical genomics research and drug discovery. BindingDB: contains a large number of known small molecules for chemical genomics research and drug discovery. SMPDB: contains a large number of known small molecules for chemical genomics research and drug discovery. In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M and Interbioscreen, which are also often used for virtual screening.

[0021] The term "affinity" as used in the present invention refers to how tightly a molecule binds to a receptor. A high affinity means that the binding is more stable, while a low affinity means that the binding is less stable. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0022] Furthermore, the method for determining the binding site of the complex further comprises:

[0023] Constructing n truncated expression mutants of various PTK6 proteins, where n is a natural number greater than or equal to 1;

[0024] Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key region for the interaction between PTK6 and hnRNPH1.

[0025] Furthermore, the key region of the interaction between PTK6 and hnRNPH1 serves as a target site for drug binding.

[0026] Furthermore, based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PTK6 and the 210th amino acid of the hnRNPH1 protein.

[0027] In some embodiments, the compound includes but is not limited to oxides, acids, bases, salts, and organic matter. In some embodiments, the oxide is a compound composed of two elements, one of which is an oxygen element. In some embodiments, the acid is a compound in which all the cations generated during ionization are hydrogen ions. In some embodiments, the base is a compound in which all the anions generated during ionization are hydroxide ions. In some embodiments, the salt is a compound that generates metal cations (or ammonium ions) and acid ions during ionization. In some embodiments, the organic matter is a general term for carbon-containing compounds (except carbon monoxide, carbon dioxide, carbonates, metal carbides, and cyanides) or hydrocarbons and their derivatives.

[0028] The present invention provides a computer-assisted drug screening system based on PTK6 and hnRNPH1, comprising:

[0029] Data acquisition unit: obtain PTK6 protein and hnRNPH1 protein data;

[0030] Site determination unit: selecting the spatial structure of the PTK6 protein and hnRNPH1 protein complex, and determining the complex binding site as the binding site of the targeted drug;

[0031] Drug screening unit: computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

[0032] The present invention provides a computer-assisted drug screening device based on PTK6 and hnRNPH1, comprising:

[0033] A processor and a memory, wherein the processor is used to call program instructions, and when the program instructions are called and executed, the computer-assisted drug screening method described above is implemented, and the memory is used to store the program instructions.

[0034] The present invention provides a computer-readable storage medium, on which a computer program is stored. When the computer program is called and executed by a processor, the computer-assisted drug screening method described above is implemented.

[0035] The term "unit" used in the present invention refers to an application specific integrated circuit (ASIC), an electronic circuit, a processor (such as a shared processor, a dedicated processor or a group processor, etc.) and memory for executing one or more software or firmware programs, a combined logic circuit and / or other suitable components supporting the described functions.

[0036] The term "device" as used in the present invention is not limited to one or a certain number of physical objects.

[0037] The term "processor" used in the present invention refers to any type of processor, including more than one processor, such as a multi-core design or multiple processors with a multi-core design for each processor. The processor can be configured to execute a sequence of computer program instructions, such as a sequence of those instructions stored in a memory, to perform various operations, processes and methods according to the exemplary embodiments of the content of the present invention.

[0038] The term "memory" as used in this disclosure refers to any type of long-term, short-term, volatile, non-volatile or other memory, and should not be limited to any specific type of memory or any specific number of memories or types of media storing memory.

[0039] The present invention provides any of the following applications.

[0040] 1) The present invention provides the use of an inhibitor of hnRNPH1 in the preparation of a drug for treating colorectal cancer.

[0041] Furthermore, the inhibitor of hnRNPH1 reduces the expression level of hnRNPH1, reduces the LLPS of hnRNPH1, reduces the phosphorylation level of hnRNPH1, reduces the acetylation level of hnRNPH1, or reduces the methylation level of hnRNPH1.

[0042] Furthermore, the inhibitor of hnRNPH1 reduces the phosphorylation level of nRNPH1.

[0043] In the present invention, an inhibitor refers to a compound that can specifically bind to hnRNPH1, especially human hnRNPH1, or to a polynucleotide or a fragment thereof that binds to hnRNPH1, and inhibits the activity or expression of the hnRNPH1 protein or polynucleotide. The inhibitor includes inhibitors of the types of proteins / protein analogs / antibodies / RNA / DNA, etc. It can be modified or unmodified. Modification generally means a change in a compound, usually in chemical form, such as the construction and removal of a chemical structure. Specific modifications include, but are not limited to, acylation (e.g., myristoylation, palmitoylation), isoprenylation or prenylation, glypiation, addition of a glycosylphosphatidylinositol (GPI) anchor, fatty acylation, addition of a flavin moiety (e.g., FMN or FAD), addition of a heme group, phosphopantetheinylation, retinyl Schiff base, glycosylation, fucosylation, alkylation, amidation, amide bond formation, hydroxylation, phosphate (O-linked) or phosphoramidite (N-linked) formation, pegylation, biotinylation, phosphorylation, carbamylation, oxidation, and combinations thereof.

[0044] Furthermore, the hnRNPH1 inhibitor reduces the phosphorylation level of the 210th amino acid of the hnRNPH1 protein.

[0045] In the present invention, LLPS of hnRNPH1 refers to the phenomenon that hnRNPH1 protein forms liquid droplets or reversible amyloid polymers in cells through a phase separation process. This process is related to the low complexity (LC) domain in the hnRNPH1 protein, especially the LC1 domain. Phase separation is an important biophysical phenomenon in cells, which allows proteins to form dynamic structures without membrane compartments in cells, thereby promoting interactions and signal transduction between biomolecules.

[0046] 2) The present invention provides the use of hnRNPH1 in screening candidate drugs for treating colorectal cancer.

[0047] 3) The present invention provides the use of an agent for inhibiting the binding between PTK6 and hnRNPH1 in the preparation of a drug for treating colorectal cancer.

[0048] Furthermore, the action site of the agent that inhibits the binding between PTK6 and hnRNPH1 is on the PTK6 protein.

[0049] Furthermore, the agent that inhibits the binding between PTK6 and hnRNPH1 is Tilfrinib.

[0050] Furthermore, the action site of the agent that inhibits the binding between PTK6 and hnRNPH1 is on the hnRNPH1 protein.

[0051] Furthermore, the action site of the agent that inhibits the binding between PTK6 and hnRNPH1 is at the 210th amino acid of the hnRNPH1 protein.

[0052] Furthermore, the reagent that inhibits the binding between PTK6 and hnRNPH1 acts on the 210th amino acid of the hnRNPH1 protein to reduce its phosphorylation level.

[0053] 4) The present invention provides the use of a complex formed by the binding of PTK6 and hnRNPH1 in screening drugs for treating colorectal cancer.

[0054] 5) The present invention provides the use of a complex formed by the binding of PTK6 and hnRNPH1 in screening agents that inhibit the binding of PTK6 and hnRNPH1.

[0055] 6) The present invention provides the use of PTK6 in preparing a regulator of the phosphorylation level of hnRNPH1 in cells.

[0056] 7) The present invention provides the use of PTK6 in preparing a cell model of hnRNPH1 hyperphosphorylation / hypophosphorylation.

[0057] Furthermore, the hnRNPH1 hyperphosphorylation / hypophosphorylation refers to the hyperphosphorylation / hypophosphorylation of the 210th amino acid of the hnRNPH1 protein.

[0058] In some embodiments, the more PTK6 binds to hnRNPH1, the higher the hnRNPH1 phosphorylation level. In some embodiments, the hnRNPH1 phosphorylation level causes a change in the LLPS degree of hnRNPH1, wherein the higher the hnRNPH1 phosphorylation level, the higher the hnRNPH1 LLPS degree.

[0059] The present invention provides a pharmaceutical composition for treating colorectal cancer, wherein the pharmaceutical composition comprises an inhibitor of hnRNPH1 and / or an agent for inhibiting the binding of PTK6 and hnRNPH1.

[0060] Furthermore, the pharmaceutical composition includes protein analogs, antibodies, and RNA drugs.

[0061] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0062] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable salt form.

[0063] In some embodiments, pharmaceutically acceptable salt forms refer to pharmaceutically acceptable salts, including salts prepared from pharmaceutically acceptable bases or acids. In some embodiments, examples of pharmaceutically acceptable bases include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, magnesium, aluminum, primary amines, secondary amines, tertiary amines, substituted amines (including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins) such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. In some embodiments, examples of pharmaceutically acceptable acids include, but are not limited to, inorganic acids and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinylsulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid.

[0064] In some embodiments, the pharmaceutically acceptable excipients include any and all solvents, diluents, buffers (e.g., neutral buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubilizers (e.g., polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavoring agents, aromatiser, thickeners, substances to obtain a storage effect, coating agents, antifungal agents, preservatives (e.g., Thimerosal™, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tonicity control agents, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol), etc. The use of such media and agents for formulating pharmaceutical compositions is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0065] The present invention provides a method for regulating the phosphorylation level of hnRNPH1 in colorectal cancer cells or tissues in vitro, and the method comprises changing the expression of PTK6 in colorectal cancer cells or tissues.

[0066] Furthermore, the method for regulating the phosphorylation level of hnRNPH1 in colorectal cancer cells or tissues comprises administering a PTK6 promoter or inhibitor to the colorectal cancer cells or tissues.

[0067] Furthermore, the method is for non-therapeutic purposes.

[0068] In some embodiments, the more PTK6 binds to hnRNPH1, the higher the hnRNPH1 phosphorylation level. In some embodiments, the hnRNPH1 phosphorylation level causes a change in the LLPS degree of hnRNPH1, wherein the higher the hnRNPH1 phosphorylation level, the higher the hnRNPH1 LLPS degree.

[0069] The present invention provides a method for constructing a hnRNPH1 hyperphosphorylation / hypophosphorylation colorectal cancer cell model, the method comprising regulating the expression of PTK6 in cells.

[0070] Furthermore, the method for constructing a colorectal cancer cell model with hyperphosphorylation of hnRNPH1 includes promoting the expression of PTK6 in the cells.

[0071] Furthermore, the promoting the expression of PTK6 in the cells comprises administering a PTK6 promoter to the cells.

[0072] Furthermore, the method for constructing a colorectal cancer cell model with hypophosphorylation of hnRNPH1 includes inhibiting the expression of PTK6 in the cells.

[0073] Further, inhibiting the expression of PTK6 in the cell comprises administering a PTK6 inhibitor to the cell.

[0074] Furthermore, the hnRNPH1 hyperphosphorylation / hypophosphorylation refers to the hyperphosphorylation / hypophosphorylation of the 210th amino acid of the hnRNPH1 protein.

[0075] In some embodiments, the more PTK6 binds to hnRNPH1, the higher the hnRNPH1 phosphorylation level. In some embodiments, the hnRNPH1 phosphorylation level causes a change in the LLPS degree of hnRNPH1, wherein the higher the hnRNPH1 phosphorylation level, the higher the hnRNPH1 LLPS degree.

[0076] In some embodiments, promoting the expression of PTK6 in cells comprises administering a promoter of PTK6 to cells; the promoter promotes the expression level of PTK6. In the present invention, a promoter refers to any substance that can increase the activity of PTK6 protein, improve the stability of PTK6 gene or protein, upregulate the expression of PTK6 protein, increase the effective action time of PTK6 protein, or promote the transcription and translation of PTK6 gene, and these substances can be used in the present invention.

[0077] In some embodiments, inhibiting the expression of PTK6 in a cell comprises administering a PTK6 inhibitor to the cell; the inhibitor inhibits the expression level of PTK6. In the present invention, an inhibitor refers to a polynucleotide or fragment thereof that can specifically bind to PTK6, or bind to PTK6, and inhibit the activity and / or expression of PTK6 protein or polynucleotide. It includes nucleic acid inhibitors, protein inhibitors, and compounds.

[0078] In this article, the PTK6 refers to the gene or protein with Gene ID: 5753 in NCBI. The hnRNPH1 (heterogeneous nuclear ribonucleoprotein H isoform c) refers to the gene or protein with Gene ID: 3187 in NCBI, and the specific amino acid code is NP_001351154.1.

[0079] Advantages and beneficial effects of the present invention:

[0080] In this study, we found that upregulated PTK6 was associated with poor prognosis and promoted autophagy while inhibiting apoptosis in colorectal cancer. Further studies showed that PTK6 phosphorylates hnRNPH1, promoting its LLPS. In addition, phase-separated hnRNPH1 affects the inclusion of NBR1 exon 10, thereby regulating the autophagic process. Our findings reveal a novel PTK6 / hnRNPH1 / NBR1 regulatory axis in autophagy, providing a potential therapeutic target for CRC. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 It is a schematic diagram of a process flow of a computer-assisted drug screening method based on PTK6 and hnRNPH1 provided by the present invention.

[0082] Figure 2 It is a schematic diagram of a computer-assisted drug screening system based on PTK6 and hnRNPH1 provided by the present invention.

[0083] Figure 3 It is a schematic diagram of a computer-assisted drug screening device based on PTK6 and hnRNPH1 provided by the present invention.

[0084] Figure 4 Figure 1 is a graph showing the expression of PTK6 in CRC and its relationship with clinicopathological features and patient prognosis. A is a Venn diagram of the intersection of CRC-related genes in the GEPIA2 and GSE138202 databases and autophagy-related genes in HADb. B is a prognostic forest diagram of PTK6, SERPINA1, NKX2-3, and BIRC5. C is the expression level of PTK6 in tumor and paracancerous tissues observed using TCGA combined with the GTEx database. D is the expression level of PTK6 in different clinical stages. E is the overall survival (OS) of PTK6 analyzed by the Kaplan-Meier (KM) method. F is the expression level of PTK6 in 53 pairs of cancer tissues and paracancerous tissues detected by Western blot. GH is the immunohistochemical score and representative images of PTK6 in tumor and paracancerous tissues in 73 pairs of tissue microarrays (TMAs). I is an analysis of the OS of patients with TMAs based on the expression level of PTK6. JK is a univariate and multivariate analysis of colorectal cancer patients in TMAs. Error bars represent mean ± SD compared with control, *p<0.05, **p<0.01, and ***p<0.001.

[0085] Figure 5The results of PTK6 inhibiting apoptosis of colorectal cancer cells by activating autophagy are shown in Figure 1. A is the transmission electron microscopy observation of autophagosomes in HCT116 and DLD1 cells. B is the Western blot analysis of the effect of PTK6 on BECN1, p62 and LC3B in HCT116. C is the immunofluorescence of LC3B in HCT116 cells under different treatments. D is the detection of changes in autophagic flux using the mCherry-EGFP-LC3 reporter experiment in HCT116. EF is the role of PTK6 in apoptosis of HCT116 cells analyzed by flow cytometry and statistics. G is the role of PTK6 in apoptosis of HCT116 cells analyzed by Western blot. HI is the effect of PTK6 on apoptosis after adding CQ (20 μM) to HCT1116 by flow cytometry and statistics. J is the effect of PTK6 on apoptosis after adding CQ (20 μM) to HCT116 analyzed by Western blot. KL is a xenograft tumor model experiment established by using HCT116 treated mice (n=5), and the tumor weight and volume were monitored during 24 days. M is immunohistochemistry for PTK6, LC3B, p62, cleaved caspase-3 and Ki67. Compared with the control group, **p<0.01, ***p<0.001.

[0086] Figure 6 The figure shows that PTK6 activates autophagy and inhibits apoptosis through hnRNPH1. A is the protein spectrum result of PTK6 immunoprecipitation separation shown by Coomassie Brilliant Blue staining. B is a Venn diagram (left) showing the intersection between PTK6 mass spectrometry results and splicing factors. By Sequest HT score prioritization, hnRNPH1 was identified as a potential interacting protein (right). CD is the immunoprecipitation of PTK6 and hnRNPH1 in HCT116 and DLD1. E is the differential splicing events associated with hnRNPH1 analyzed by RNA-seq, and then the genes involved in these alternative splicing events were identified by KEGG analysis. F is the detection of changes in autophagic flux using mCherry-EGFP-LC3 reporter experiments in HCT116. G is a Western blot analysis of the effect of hnRNPH1 on BECN1, p62 and LC3B in HCT116. HI is the effect of hnRNPH1 on cell apoptosis after the addition of CQ (20μM) in HCT1116 by flow cytometry and statistical analysis. J is flow cytometry analysis of HCT116, control group, sh-PTK6 group and sh-PTK6+hnRNPH1 OEK is the flow cytometry analysis of HCT116 cell apoptosis. L is the Western blot method to detect the expression of BECN1, p62, LC3B, cleaved caspase-3, BAX, and Bcl2 proteins in HCT116 cells, and HCT116 cells were divided into control group, sh-PTK6 group, and sh-PTK6+hnRNPH1 group. OE Group M is the analysis of the changes in autophagic flux in HCT116 cells using the mCherry-EGFP-LC3 reporter assay, comparing three groups: control group, sh-PTK6, and sh-PTK6+hnRNPH1 OE Compared with the control group, **p<0.01, ***p<0.001.

[0087] Figure 7 The figure is the result of PTK6 phosphorylating hnRNPH1 at the Y210 site. Among them, A is the Phos-tag SDS-PAGE analysis (upper figure) showing the effect of PTK6 on hnRNPH1 phosphorylation, while the conventional SDS-PAGE analysis (lower figure) has no band at the same position. B is the prediction of phosphorylation sites on hnRNPH1 using PhosphoSitePlus and NetPhos 3.1 tools. C is a schematic diagram of wild-type (WT) and mutant (tyrosine replaced by alanine) hnRNPH1 protein. DE is the use of Western blot analysis of the effect of PTK6 on various hnRNPH1 mutants in HCT116 and DLD1. F is an in vitro phosphorylation experiment to explore the effect of PTK6 on various hnRNPH1 mutants. G is a truncation model diagram of PTK6 protein. H is a Western blot analysis of the IP experiment of HCT116 and DLD1, showing the interaction between PTK6 and hnRNPH1.

[0088] Figure 8 The results of the in vivo and in vitro phase separation ability of hnRNPH1 are shown in Figure 1. A shows the phase separation of EGFP-hnRNPH1 and EGFP-hnRNPH1 observed under fluorescence and bright field vision. Y210AImages in HCT116. B is a three-dimensional reconstruction image of EGFP-hnRNPH1 living cells observed in HCT116 using a laser scanning confocal microscope (CLSM, Leica STELLARIS 5, Germany). C is an image of two EGFP-hnRNPH1 droplets in HCT116 merging to form a larger droplet. D is an image of EGFP-hnRNPH1 droplets exposed to 10% 1,6-hexanediol and then recovered after the removal of 1,6-hexanediol. E is an image (left) and quantification (right) of EGFP-hnRNPH1 FRAP in HCT116. Data are expressed as mean ± standard deviation. (n = 3 drops). F is a representative fluorescence image showing the phase separation behavior of purified EGFP-hnRNPH1 at different NaCl concentrations and the addition of 5% PEG8000. G is a three-dimensional reconstruction image of two in vitro purified EGFP-hnRNPH1 droplets merging to form a larger droplet. H is a three-dimensional reconstruction image of in vitro purified EGFP-hnRNPH1 observed using a laser scanning confocal microscope. I is the FRAP image (left) and quantification (right) of in vitro purified EGFP-hnRNPH1. Data are shown as mean ± SD (n = 3 droplets). J is the IF analysis (left) and quantification (right) of hnRNPH1 puncta in CRC and normal tissues. K is the IF analysis (left) and quantification (right) of hnRNPH1 puncta in primary cell lines of CRC and normal colon epithelial tissues. **p < 0.01, ***p < 0.001.

[0089] Fig. 9 The results of PTK6 regulating the phase separation ability of hnRNPH1 by phosphorylating Y210. A is the analysis of the disordered region of hnRNPH1 by IUPred2A. B is a schematic diagram of four plasmids of different hnRNPH1 variants labeled with EGFP. C is a confocal microscopy image of cells transfected with different hnRNPH1 plasmids in HCT116. DE is HCT116 cell EGFP-hnRNPH1 and EGFP-hnRNPH1 △206-246 FRAP images and quantitative analysis. Data are presented as mean ± SD (n = 3 droplets). FG is EGFP-hnRNPH1, EGFP-hnRNPH1 Y210A and EGFP-hnRNPH1 IDR Images and quantification of FRAP, and FRAP after PTK6 overexpression in HCT116. Data are presented as mean ± SD (n = 3 droplets).

[0090] Fig.10The following is a diagram showing the effect of the PTK6 / hnRNPH1 / NBR1 axis on autophagy and apoptosis, as well as a diagram showing the mechanism of promoting the retention of NBR1 exon 10. A is a flowchart for screening candidate target genes for hnRNPH1. B is a salmon diagram for analyzing alternative splicing transcripts of NBR1 in HCT116 using rMATS. C is a semi-quantitative PCR analysis of the percentage of NBR1 exon 10+ transcripts in HCT116 after hnRNPH1 knockdown and overexpression. D is a semi-quantitative PCR analysis of the expression of EGFP-hnRNPH1 and EGFP-hnRNPH1 △206-246 The percentage of NBR1 exon 10+ transcripts in HCT116. E is the percentage of NBR1 exon 10+ transcripts in HCT116 of the five experimental groups analyzed by semi-quantitative PCR. F is the IF of hnRNPH1, SRRM2 and SON in HCT116 and DLD1. G is a sequence diagram (top) showing the preferred binding sequence of hnRNPH1, and primers designed for potential binding regions (bottom). H is CLIP-qPCR analysis of the association between hnRNPH1 and NBR1 pre-mRNA at the indicated positions. I is the NBR1 minigene construction diagram (NBR1 WT and NBR1 ΔExon10 JP for Western blot analysis, mCherry-EGFP-LC3 dual fluorescence reporter experiment and flow cytometry detection of NBR1 WT and NBR1 ΔExon10 Effects of PTK6 on autophagy and apoptosis in HCT116 and DLD1 cells. Bars represent mean ± SD; ns indicates no significant difference, ***p < 0.001.

[0091] Fig.11 This is an experimental result showing that Tilfrinib inhibits PTK6-activated autophagy and promotes cell apoptosis in in vitro and in vivo experiments.

[0092] Among them, A is the molecular structure of Tilfrinib. B is the Western blot analysis of the expression of BECN1, p62 and LC3B after different concentrations of Tilfrinib acted on HCT116 for 24 h. C is the electron microscopy observation of the changes in autophagosomes in HCT116 and DLD1 after Tilfrinib treatment. D is the mCherry-EGFP-LC3 reporter experiment used to study the effect of Tilfrinib on PTK6-mediated autophagic flux in HCT116. E is the flow cytometry detection of the effect of Tilfrinib on PTK6-mediated apoptosis of HCT116 cells. F is the Western blot analysis of the expression of BECN1, p62, LC3B, cleaved caspase-3, BAX and Bcl2 in HCT116 cells to explore the effect of Tilfrinib on HCT116 cells. G is the tumor growth of patient-derived CRC organoids treated with Tilfrinib (20 μM). HJ: HCT116 was used to establish a mouse xenograft tumor model (n=5), and the tumor weight and volume were monitored during 24 days. The treatment group was intraperitoneally injected with Tilfrinib (10 mg / kg) once every 3 days. K: Immunohistochemistry for PTK6, LC3B, p62, Ki67, and cleaved caspase-3. The rightmost column shows the NBR1 exon 10 specific probe. ***p<0.001.

[0093] Fig.12 This is a schematic diagram of the molecular mechanism of autophagy mediated by the novel PTK6 / hnRNPH1 / NBR1 regulatory axis.

[0094] Fig.13 Bioinformatics analysis of PTK6 expression and its relationship with autophagy and apoptosis Figure 1 . Among them, A is the expression level of PTK6 in CRC evaluated by GEPIA2 analysis. B is the expression level of PTK6 in 53 pairs of cancer tissues and adjacent tissues detected by qRT-PCR. C is the DFS analysis of TMA patients based on PTK6 expression level. D is the relationship between PTK6 and autophagy process analyzed by GSEA. Bars represent mean ± standard deviation; *p<0.05 and **p<0.01.

[0095] Fig.14Figure 1 is a graph showing that PTK6 inhibits apoptosis in CRC by activating autophagy. A is the evaluation of mRNA and protein expression levels of PTK6 in different cell lines by Western blot and qPCR analysis. B is the Western blot analysis of the effect of PTK6 on BECN1, p62, and LC3B in DLD1. C is the IF of LC3B in DLD1 under different treatments. D is the detection of changes in autophagic flux using the mCherry-EGFP-LC3 reporter experiment in DLD1. EF is the analysis of the role of PTK6 in apoptosis of DLD1 cells by flow cytometry and statistics. G is the analysis of the role of PTK6 in apoptosis of DLD1 cells by Western blot. HI is the analysis of the effect of PTK6 on apoptosis after the addition of CQ (20 μM) to DLD1 by flow cytometry and statistics. J is the analysis of the effect of PTK6 on apoptosis after the addition of CQ (20 μM) to DLD1 by Western blot. KL is the detection of apoptosis of transplanted tumor tissue cells by TUNEL method and statistical analysis. M is the TUNEL method to detect the apoptosis of HCT116 and DLD1 cells after CQ (20 μM) treatment, and statistical analysis was performed. N is the flow cytometry analysis of the apoptosis of HCT116 and DLD1 cells treated with CQ (20 μM), and the apoptotic cell population was counted. O is the Western blot analysis of the expression of cleaved caspase-9, caspase-3, cleaved caspase-3, cleaved caspase-8, BAX and Bcl2 in HCT116 and DLD1 treated with CQ (20 μM). P is the Western blot analysis of the expression of cleaved caspase-8 and p62 in HCT116 and DLD1. Bars represent mean ± standard deviation; **p<0.01 and ***p<0.001.

[0096] Fig.15 This is a diagram of PTK6 activating autophagy and inhibiting apoptosis through hnRNPH1. Among them, A is the KEGG enrichment analysis of genes identified by PTK6-IP-MS. B is the RNA-seq analysis of sh-NC and sh-hnRNPH1 samples. C is the detection of changes in autophagic flow in DLD1 cells using the mCherry-EGFP-LC3 reporter experiment. D is a Western blot analysis of the effect of hnRNPH1 on BECN1, p62 and LC3B in DLD1. EF is a flow cytometric analysis of the effect of hnRNPH1 on apoptosis after the addition of CQ (20μM) to DLD1. G is a Western blot analysis of DLD1 divided into 3 groups: control group, sh-PTK6 group and sh-PTK6+hnRNPH1 OEThe expression of BECN1, p62, LC3B, cleaved caspase-3, BAX and Bcl2 in the DLD1 cells were detected to evaluate the effect of DLD1 on autophagy and apoptosis. H was used to analyze the changes in autophagic flux in DLD1 cells using the mCherry-EGFP-LC3 reporter assay, comparing the three groups: control group, sh-PTK6 group and sh-PTK6+hnRNPH1 OE IJ shows the apoptosis of cells in the three experimental groups in DLD1 detected by flow cytometry. The bars represent mean ± standard deviation; **p<0.01 and ***p<0.001. Fig.16 The results of PTK6 regulating the phase separation ability of hnRNPH1 by phosphorylating Y210. Among them, A is the observation of EGFP-hnRNPH1 and EGFP-hnRNPH1 under fluorescence and bright field microscopes Y210A Expression in DLD1. B is a three-dimensional reconstruction image of living cells expressing EGFP-hnRNPH1 in DLD1 observed using a confocal laser scanning microscope (CLSM, Leica STELLARIS 5, Germany). C is two EGFP-hnRNPH1 droplets fused in DLD1 to form a larger droplet. DF are images of EGFP-hnRNPH1 droplets exposed to 10% 1,6-hexanediol and then recovered after the removal of 1,6-hexanediol. G is an image (top) and quantification (bottom) of FRAP of EGFP-hnRNPH1 in DLD1. Data are expressed as mean ± standard deviation. (n = 3 droplets). HI is a confocal microscopy image of cells transfected with different hnRNPH1 plasmids. J is an image of EGFP-hnRNPH1 and EGFP-hnRNPH1 in DLD1. △206-246 FRAP images and quantitative analysis. Data are presented as mean ± SD (n = 3 droplets). KL is EGFP-hnRNPH1, EGFP-hnRNPH1 Y210A and EGFP-hnRNPH1 IDR Images and quantification of FRAP, and FRAP after PTK6 overexpression in DLD1. Data are presented as mean ± SD (n = 3 droplets). Bars represent mean ± SD; ns indicates no significant difference, **p < 0.01 and ***p < 0.001.

[0097] Fig.17PTK6 promotes LLPS of hnRNPH1, mediates retention of NBR1 exons, thereby activating autophagy and inhibiting apoptosis. Result figure. A is an analysis of different alternative splicing events regulated by hnRNPH1, including SE (exon skipping), A3SS (variable 3' splicing site), A5SS (variable 5' splicing site), RI (intron retention) and MXE (mutually exclusive exons). B is the proportion of alternative splicing events regulated by hnRNPH1. C is the percentage of NBR1 exon 10 + transcripts in DLD1 after hnRNPH1 knockdown using semi-quantitative PCR analysis. D is the expression of EGFP-hnRNPH1 and EGFP-hnRNPH1 using semi-quantitative PCR analysis. △206-246 The percentage of NBR1 exon 10+ transcripts in DLD1 of the five experimental groups was analyzed by semi-quantitative PCR. F was the expression of BECN1, p62, LC3B, cleaved caspase-3, BAX and Bcl2 detected by Western blot in HCT116 and DLD1 cells to evaluate the expression of NBR1 WT and NBR1 ΔExon10 Effects on autophagy and apoptosis. G is the observation of NBR1 by mCherry-EGFP-LC3 dual fluorescence reporter experiment WT and NBR1 ΔExon10 Effects on autophagic flux in HCT116 and DLD1 cells. H is an mCherry-EGFP-LC3 dual fluorescence reporter assay to detect changes in autophagic flux and evaluate the effect of NBR1 WT and NBR1 ΔExon10 Effects of PTK6 on DLD1 autophagy. I is the observation of NBR1 by flow cytometry WT and NBR1 ΔExon10 Effects on apoptosis of HCT116 and DLD1 cells. J is the observation of NBR1 in HCT116 and DLD1 cells using a confocal laser scanning microscope (CLSM, Leica STELLARIS 5, Germany). WT and NBR1 ΔExon10 K is a schematic diagram of the truncated form of NBR1 protein. L is a Western blot analysis of the IP experiment of HCT116 and DLD1, showing the interaction between NBR1 and p62. M is a CO-IP detection of NBR1 in HCT116 and DLD1 WT and NBR1 ΔExon10 Interaction of N with p62 in HCT116 and DLD1 by confocal microscopy WT and NBR1 ΔExon10Changes of p62 after treatment. Bars represent mean ± SD; Ns indicate no significant difference, *p < 0.05 and ***p < 0.001.

[0098] Fig.18 Figure 1 shows the results of Tilfrinib inhibiting PTK6-activated autophagy, thereby promoting cell apoptosis. A is an mCherry-EGFP-LC3 reporter experiment to study the effect of Tilfrinib on PTK6-mediated autophagic flux in DLD1. BD is a flow cytometry assay to detect the effect of Tilfrinib on PTK6-mediated apoptosis of DLD1 and HCT116 cells. E is a Western blot analysis of the expression of BECN1, p62, LC3B, cleaved caspase-3, BAX, and Bcl2 in DLD1 to explore the effect of Tilfrinib on DLD1. F is a statistical analysis of the width of patient-derived CRC organoids in different groups. GH is a TUNEL method to detect apoptosis of transplanted tumor tissue cells and perform statistical analysis. I is a laser scanning confocal microscope (CLSM, Leica STELLARIS 5, Germany) to observe the changes in LLPS in HCT116 and DLD1 after Tilfrinib treatment. DETAILED DESCRIPTION

[0099] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0100] Example 1 Elevated expression of PTK6 in colorectal cancer is associated with clinical pathological features and patient prognosis

[0101] To investigate the role of autophagy in the development of CRC, we used the Human Autophagy Database (HADb, http: / / www.autophagy.lu / ) to identify 222 autophagy-related genes. We then screened for CRC upregulated genes in the GEPIA2 database and the public dataset GSE138202. By integrating these data, we found four key genes: PTK6, SERPINA1, NKX2-3, and BIRC5 ( Figure 4 A). The relationship between PTK6 and clinical prognosis was further analyzed by univariate Cox regression, and it was found that PTK6 showed a higher degree of malignancy compared with other genes ( Figure 4 B).

[0102] The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets as well as the GEPIA2 database (http: / / gepia.cancer-pku.cn / ) showed that PTK6 was significantly upregulated in colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). Figure 4 C and Fig.13 A). Kaplan-Meier (KM) analysis showed that patients with high PTK6 expression had a poor prognosis, and its expression was correlated with clinical stage ( Figure 4 DE).

[0103] Subsequently, we detected the expression of PTK6 in 53 pairs of fresh colorectal cancer and adjacent normal tissue samples. The results showed that compared with normal tissues, the protein and mRNA expression levels of PTK6 in colorectal cancer tissues were significantly increased (p<0.001) ( Figure 4 F and Fig.13 B). In addition, we performed immunohistochemistry (IHC) analysis on a tissue microarray (TMA) containing 73 tissue pairs ( Figure 4 G). The results showed that the PTK6 IHC score of tumor tissue was significantly higher than that of adjacent normal tissue ( Figure 4 H).

[0104] To further explore the clinical significance of PTK6 in the TMA cohort, we used the chi-square test to analyze the relationship between PTK6 expression and various clinicopathological characteristics. The results showed that high PTK6 expression was positively correlated with tumor size (p = 0.001), invasion depth (p < 0.001), lymph node metastasis (p = 0.011), distant metastasis (p = 0.017) and TNM stage (p = 0.006) (Table 1). KM survival curve and Cox regression analysis were used to evaluate the prognostic value of PTK6 in colorectal cancer. KM analysis showed that patients with high PTK6 expression had poorer overall survival (OS) and disease-free survival (DFS) compared with patients with low PTK6 expression (p = 0.0012; p = 0.001)( Figure 4 I and Fig.13 C). Univariate Cox regression analysis showed that PTK6 expression, TNM stage, lymph node metastasis and distant metastasis were significant risk factors affecting the prognosis of CRC patients ( Figure 4 J). In addition, multivariate Cox regression analysis confirmed that PTK6 expression and TNM stage were independent prognostic factors ( Figure 4 K).

[0105] Table 1 Correlation between PTK6 expression in TMAs and clinicopathological features

[0106]

[0107]

[0108] At the same time, we performed gene set enrichment analysis (GSEA) on the gene expression matrix of CRC patients obtained from the TCGA database to compare the PTK6 high expression group and low expression group. The GSEA results showed that PTK6 expression was positively correlated with the autophagy regulation pathway ( Fig.13 D) PTK6 is highly expressed in colorectal cancer tissues and is associated with the clinicopathological features and prognosis of colorectal cancer patients.

[0109] Example 2 PTK6 inhibits apoptosis of colorectal cancer by activating autophagy both in vitro and in vivo

[0110] To explore the biological function of PTK6 in colorectal cancer, we preliminarily analyzed its expression in different cell lines. Our results showed that the expression of PTK6 in HCT116 was the most significantly increased, while the increase in DLD1 was the least significant ( Fig.14 A). Therefore, we selected these two cell lines for subsequent PTK6 knockdown and overexpression experiments. We constructed two shRNAs targeting PTK6 (sh-PTK6 #1 ,sh-PTK6 #2 ) and a PTK6 overexpression vector (PTK6 OE ). Among them, sh-PTK6 #1 The sequence is: CATCCATGGTTAAGTCATA. sh-PTK6 #2 The sequence is: TGAAGAAGCTGCGGCACAA.

[0111] Transmission electron microscopy (TEM) showed that the number of autophagosomes increased significantly after PTK6 overexpression in HCT116 and DLD1 cell lines ( Figure 5 A). Western blot analysis further showed that PTK6 overexpression led to a significant increase in the LC3B-II / LC3B-I ratio and BECN1 levels, accompanied by a decrease in p62 expression, indicating enhanced autophagic flux. In contrast, PTK6 knockdown resulted in decreased autophagic activity ( Figure 5 B and Fig.14 B). Immunofluorescence staining of LC3B further revealed that high expression of PTK6 significantly enhanced the expression level of LC3B ( Figure 5 C and Fig.14C). To further evaluate the role of PTK6 in autophagy, we used an mCherry-GFP-LC3 reporter assay to assess changes in autophagic flux in the presence of chloroquine (CQ), a lysosomal inhibitor that blocks autophagic degradation. The data showed that PTK6 enhanced autophagy in the absence and presence of CQ ( Figure 5 D and Fig.14 D).

[0112] Flow cytometry and Western blot analysis showed that PTK6 overexpression significantly inhibited cell apoptosis, while PTK6 knockdown had the opposite effect ( Figure 5 EG and Fig.14 EG). In addition, flow cytometry and Western blot confirmed that PTK6 inhibits apoptosis by promoting autophagy ( Figure 5 HJ and Fig.14 HJ).

[0113] To further verify the function of PTK6 in vivo, we performed xenograft tumor model experiments in mice and obtained tumors for IHC after 24 days. We constructed stable cell lines with PTK6 knockdown or overexpression, and then injected these cell lines subcutaneously into nude mice. Two independent experimental groups and their corresponding control groups were established for comparison. Tumor volume was measured regularly. The results showed that PTK6 significantly promoted tumor growth, and the tumor volume and weight were significantly higher than those in the control group (p<0.001)( Figure 5 K). In contrast, PTK6 knockdown resulted in a significant decrease in tumor growth (p<0.001) ( Figure 5 L). TUNEL and IHC analysis further confirmed our in vitro findings, indicating that PTK6 directly enhanced autophagy and simultaneously inhibited apoptosis of tumor cells ( Figure 5 M and Fig.14 KL).

[0114] Previous studies have shown that under protective autophagy, most of the cleaved caspase-8 is sequestered by autophagosomes and subsequently degraded by lysosomes. To investigate whether autophagy inhibits apoptosis in CRC by selectively degrading pro-apoptotic proteins, we used CQ to block autophagic lysosomal degradation. Apoptosis of CRC cells after CQ treatment was assessed by flow cytometry and TUNEL assay ( Fig.14 MN). The results showed that blocking autophagic lysosomal degradation significantly induced apoptosis in CRC cells. In addition, we analyzed the expression levels of cleaved caspase-9, caspase-3, cleaved caspase-8, BAX, and Bcl2 in CQ-treated CRC cells ( Fig.14O). We observed that CQ treatment increased the expression of cleaved caspase-3 and cleaved caspase-8, but had no significant effect on the level of cleaved caspase-9. Then, Western blot results showed that PTK6-mediated autophagy reduced the expression of cleaved caspase-8, thereby inhibiting CRC apoptosis ( Fig.14 P).

[0115] Together, these findings suggest that PTK6 enhances autophagy and subsequently inhibits apoptosis in both in vitro and in vivo models. Furthermore, PTK6-mediated autophagy selectively degrades cleaved caspase-8, effectively blocking apoptosis.

[0116] Example 3 PTK6 activates autophagy through hnRNPH1 and inhibits cell apoptosis

[0117] To further elucidate the role of PTK6 in promoting autophagy, we used immunoprecipitation-mass spectrometry (IP-MS) technology ( Figure 6 A) 1,363 potential PTK6-binding proteins were identified. KEGG analysis showed that PTK6 was enriched in the spliceosome pathway ( Fig.15 A). As a result, we identified 53 proteins belonging to different key splicing factor families, including the serine / arginine-rich splicing factor (SRSF) family, the heterogeneous nuclear ribonucleoprotein (hnRNP) family, the U small nuclear ribonucleoprotein (UsnRNP) family, the polypyrimidine tract binding protein (PTB) family, the poly(rC) binding protein (PCBP) family, and other important splicing factors. By integrating IP-MS data and splicing factor information, we identified 19 splicing factors associated with PTK6. Among them, hnRNPH1, which had the highest Sequest HT score, was selected as the most reliable splicing factor ( Figure 6 B). Co-immunoprecipitation (Co-IP) assay further validated the interaction between PTK6 and hnRNPH1 ( Figure 6 CD).

[0118] To determine the function of hnRNPH1, we performed RNA-seq in HCT116 cells transfected with sh-hnRNPH1 and sh-NC. Fig.15 B) Among them, sh-hnRNPH1 #1 The sequence is CCGGGATCCACCACGAAAG. sh-hnRNPH1 #2 The sequence is CGGAGCTGAAGTTAGAACTCA. It is worth noting that KEGG analysis showed that hnRNPH1 was significantly associated with autophagy and apoptosis ( Figure 6E). To further validate the role of hnRNPH1 in autophagy, we performed mCherry-EGFP-LC3 dual fluorescence reporter experiments to demonstrate that hnRNPH1 increased autophagic flux ( Figure 6 F and Fig.15 C). This observation was confirmed by Western blot ( Figure 6 G and Fig.15 D). Subsequently, we used flow cytometry to evaluate the role of hnRNPH1 in apoptosis. After adding CQ, we found that hnRNPH1 promoted autophagy and then inhibited apoptosis ( Figure 6 HI and Fig.15 EF). To investigate the relationship between PTK6 and hnRNPH1 in regulating autophagy and apoptosis, we used Western blot, mCherry-EGFP-LC3 dual fluorescence reporter assay, and flow cytometry ( Figure 6 JM and Fig.15 GJ). These results indicate that hnRNPH1 overexpression can rescue the effects of PTK6 knockdown on autophagy and apoptosis. Taken together, these findings suggest that PTK6 may promote autophagy and inhibit apoptosis through the action of hnRNPH1.

[0119] Example 4 PTK6 phosphorylates hnRNPH1 at Y210

[0120] To investigate the potential mechanism, we first conducted Mn 2+ -Phos-tag SDS-PAGE experiment was performed to verify whether protein tyrosine kinase PTK6 can phosphorylate hnRNPH1, thereby promoting its biological function in CRC. The results showed that overexpression of PTK6 significantly increased the phosphorylation level of hnRNPH1, while knocking down PTK6 produced the opposite effect ( Figure 7 A). Using PhosphoSitePlus (https: / / www.phosphosite.org) and NetPhos3.1

[0121] (https: / / services.healthtech.dtu.dk / services / NetPhos-3.1 / ) identified 19 potential phosphorylation sites and 8 tyrosine phosphorylation sites on hnRNPH1, of which 4 tyrosine phosphorylation sites overlapped (Y210, Y253, Y276, Y306). Figure 7 B). Therefore, we mutated the four potential phosphorylation sites of hnRNPH1, Y210, Y253, Y276, and Y306, to alanine, thereby generating Y210A, Y253A, Y276A, and Y306A mutants ( Figure 7C) Then, we added PTK6 OE Co-transfected with various hnRNPH1 mutants. After transfection, we purified hnRNPH1 protein using IP and then incubated the purified hnRNPH1 with a pan-tyrosine phosphorylation antibody to assess the phosphorylation level. Western blot analysis showed that co-transfected PTK6 OE and hnRNPH1 WT Interestingly, the phosphorylation level of hnRNPH1 was significantly increased in the three mutants (hnRNPH1 Y253A hnRNPH1 Y276A and hnRNPH1 Y306A ) does not affect PTK6-mediated hnRNPH1 phosphorylation ( Figure 7 D, E). In the case of co-transfection with hnRNPH1 Y210A In cells with PTK6, the effect of PTK6 on hnRNPH1 phosphorylation disappeared, indicating that Y210 is a key site for PTK6-mediated hnRNPH1 phosphorylation ( Figure 7 DE). In addition, in vitro kinase assays confirmed that Y210 is an essential site for PTK6-mediated hnRNPH1 phosphorylation, while mutations at Y253, Y276, and Y306 sites did not affect its phosphorylation level ( Figure 7 F).

[0122] As mentioned above, PTK6 consists of three domains: SH3, SH2, and the tyrosine kinase catalytic domain (TyrKc). To identify the domains involved in the binding of PTK6 to hnRNPH1, we constructed a series of Flag-tagged truncated PTK6 proteins, including the full-length PTK6 WT PTK6 ΔSH2 (lacking the SH2 domain) and PTK6 ΔSH3 (lacking SH3 domain)( Figure 7 G). Immunoprecipitation and Western blot results showed that PTK6 WT and PTK6 ΔSH3 Both significantly bind to hnRNPH1, while PTK6 ΔSH2 Unable to bind to hnRNPH1 ( Figure 7 H).

[0123] Combined with these results and the phosphorylation mechanism of PTK6, these findings suggest that PTK6 binds to hnRNPH1 through its SH2 domain and mediates phosphorylation of hnRNPH1 Y210 through its TyrKc.

[0124] Example 5hnRNPH1 exhibits LLPS ability both in vitro and in vivo

[0125] Phosphorylation is an important post-translational modification (PTM) that significantly affects protein localization by adding phosphate groups to serine, threonine or tyrosine residues. WT and EGFP-hnRNPH1 Y210A After transfection, both bright field and fluorescence microscopy showed that hnRNPH1 formed droplet-like structures, and in the presence of EGFP-hnRNPH1 Y210A A significant reduction in the number of droplets observed in Figure 8 A and Fig.16 A).

[0126] To confirm the LLPS phenomenon of hnRNPH1, we noticed that EGFP-hnRNPH1 formed droplet-like structures in both HCT116 and DLD1 cell lines, and individual droplets merged into larger droplets ( Figure 8 BC and Fig.16 BC). Fluorescence recovery after photobleaching (FRAP) further demonstrated that EGFP-hnRNPH1 exhibited liquid-like properties ( Figure 8 E and Fig.16 G). To verify these observations, we introduced 1,6-hexanediol (1,6-HD) as an LLPS inhibitor. Existing studies have shown that 1,6-HD inhibits LLPS by interfering with weak hydrophobic interactions between proteins or between proteins and RNA. In this experiment, after treatment with 1,6-HD, the number of hnRNPH1 droplets was significantly reduced or even disappeared. After removing 1,6-HD, the droplets gradually re-formed ( Figure 8 D and Fig.16 DF).

[0127] We purified His-tagged EGFP-hnRNPH1 to investigate whether hnRNPH1 undergoes LLPS in vitro. The results showed that the purified protein formed droplet-like punctate structures in salt solution, and the number of droplets decreased with increasing salt concentration ( Figure 8 F, Figure 8 H). In addition, the number of hnRNPH1 droplets was positively correlated with protein concentration ( Figure 8 F). Observations from in vitro experiments and FRAP analysis confirmed that these droplets exhibit dynamic liquid properties ( Figure 8 G, Figure 8 I).

[0128] To determine whether there is a difference in the phase separation of hnRNPH1 in CRC and normal tissues, we used tissue sections and IF experiments to investigate the number of hnRNPH1 droplets in CRC and normal tissues. The results showed that hnRNPH1 droplets increased in CRC tissues compared with normal tissues ( Figure 8 J). In addition, we established primary cell lines from CRC and normal colon epithelial tissues and performed IF experiments. The results showed that hnRNPH1 droplets were increased in CRC primary cells compared with normal colon epithelial primary cells ( Figure 8 K). These findings may be due to the fact that high expression of PTK6 mediates increased hnRNPH1 phosphorylation, leading to a different phase separation state.

[0129] In conclusion, our results indicate that hnRNPH1 can undergo phase separation both in vitro and in vivo, which may be associated with PTK6 phosphorylation.

[0130] Example 6 PTK6 regulates the LLPS ability of hnRNPH1 by phosphorylating Y210

[0131] To investigate the relationship between LLPS and phosphorylation, we first identified the IDR of hnRNPH1 using IUPred2A (https: / / iupred2a.elte.hu / ) ( Fig. 9 A). Notably, we found that Y210 is located within the IDRs region. To evaluate the effect of Y210 on LLPS, we constructed four plasmids encoding different variants of EGFP-tagged hnRNPH1: full-length hnRNPH1 protein (EGFP-hnRNPH1), a deletion mutant lacking amino acids 206-246 within the LC1 domain,

[0132] (EGFP-hnRNPH1 Δ206-246 ), a point mutant in which tyrosine 210 in the LC1 domain was replaced by alanine (EGFP-hnRNPH1 Y210A ), and the IDRs of hnRNPA1 were added to EGFP-hnRNPH1 Y210A The mutant (EGFP-hnRNPH1 IDR )( Fig. 9 B) After cell transfection, we found that EGFP-hnRNPH1 Δ206-246 and EGFP-hnRNPH1 Y210A The LLPS of α was weakened, while the addition of IDRs restored the droplet formation ( Fig. 9 C and Fig.16 In addition, in vivo observations and FRAP experiments showed that hnRNPH1 lacking IDRs exhibited weakened phase separation ability and prolonged recovery time ( Fig. 9 DE and Fig.16 J).

[0133] To further investigate the ability of PTK6 to regulate LLPS of hnRNPH1 via phosphorylation of Y210, we performed several FRAP experiments ( Fig. 9 FG and Fig.16 KL). The results showed that EGFP-hnRNPH1 Y210A The prolonged fluorescence recovery time indicated that the mutation had an adverse effect on the LLPS ability of hnRNPH1 ( Fig. 9 FG and Fig.16 KL). Notably, when co-expressed with PTK6, there was only a small change in fluorescence recovery, further indicating that phosphorylation at the Y210 site is critical for the regulation of hnRNPH1 LLPS ( Fig. 9 FG and Fig.16 KL). In addition, in the transfection of EGFP-hnRNPH1 IDR hnRNPH1 restored its LLPS ability, while PTK6 did not affect this process ( Fig. 9 FG and Fig.16 These results suggest that PTK6 regulates the LLPS ability of hnRNPH1 by phosphorylating Y210.

[0134] Example 7 PTK6 promotes hnRNPH1 LLPS and NBR1 exon retention, thereby activating autophagy and inhibiting apoptosis

[0135] To further elucidate the mechanism by which the PTK6 / hnRNPH1 axis drives CRC autophagy, we performed hnRNPH1-RNA-seq analysis. Given the role of hnRNPH1 in RNA splicing, we analyzed its alternative splicing events, a key mechanism for generating multiple mRNA variants and protein isoforms ( Fig.17 A). RNA-seq detected a total of 81,178 alternative splicing events associated with hnRNPH1, including 60,280 exon skipping (SE) events, 12,400 mutually exclusive exon (MXE) events, 929 intron retention (IR) events, 4,757 alternative 3' splice site (A3SS) events, and 2,812 alternative 5' splice site (A5SS) events ( Fig.17 B) We focused on the major splicing events regulated by hnRNPH1, the SE events.

[0136] First, we screened for SE events regulated by hnRNPH1 (n = 16,375; p < 0.05) ( Fig.10A). By integrating the data of key autophagy genes, we identified 83 related genes. Among them, 14 genes were regulated by hnRNPH1 through SE events. Finally, we screened out the highest-scoring SE event based on the inclusion level difference score, which was exon 10 skipping of NBR1. In addition, we used rMATS to perform differential alternative splicing analysis, and the results showed that hnRNPH1 knockdown significantly increased exon 10 skipping of NBR1 ( Fig.10 B). In addition, we designed primers to investigate the effect of hnRNPH1 on this splicing event ( Fig.10 C and Fig.17 C). The RT-PCR verification results were consistent with the sequencing results.

[0137] To further determine whether LLPS affects alternative splicing events, RT-PCR analysis showed that hnRNPH1 promoted the inclusion of NBR1 exon 10, while EGFP-hnRNPH1 Δ206-246 Mutants do not ( Fig.10 D and Fig.17 D) In ​​addition, we performed co-transfection experiments to verify the role of the PTK6 / hnRNPH1 axis in NBR1 alternative splicing. The results showed that PTK6 knockdown increased NBR1 exon 10 skipping. WT Overexpression rescued its effect on NBR1 splicing ( Fig.10 E and Fig.17 E) In contrast, mutant hnRNPH1 Y210A Restoration of LLPS function of hnRNPH1 also rescued the skipping of exon 10 of NBR1. Fig.10 E and Fig.17 E) These results suggest that PTK6 promotes LLPS of hnRNPH1 through phosphorylation at Y210, thereby regulating alternative splicing of NBR1.

[0138] Nuclear speckles are known to be important membraneless organelles for mRNA splicing. Previous studies have shown that SRRM2 and SON are involved in the formation of nuclear speckles. Therefore, we used these two proteins as markers for nuclear speckles. IF experiments showed that hnRNPH1, a splicing factor, colocalized with SRRM2 and SON ( Fig.10 F). To identify the hnRNPH1 binding motif in NBR1 exon 10, we used Jalview software to perform motif analysis on all SE events and found that hnRNPH1 seemed to prefer a specific sequence pattern ( Fig.10G). To investigate the binding sequence of hnRNPH1 in NBR1 pre-mRNA, we performed in vivo cross-linking followed by immunoprecipitation and RT-PCR assays ( Fig.10 H). Our results showed that hnRNPH1 mainly binds to sequences in the region near exon 10 of NBR1.

[0139] We further constructed wild-type NBR1 (NBR1 WT ) and NBR1 with exon 10 deletion (NBR1 ΔExon10 )Plasmid( Fig.10 I) mCherry-EGFP-LC3 dual fluorescence reporter experiments, flow cytometry and western blot analysis showed that NBR1 WT Significantly promoted autophagic flux, whereas NBR1 lacking exon 10 was unable to accomplish this function ( Fig.17 F, Fig.17 G, Fig.17 I). In addition, NBR1 WT It can rescue the effects of PTK6 on autophagy and apoptosis, while NBR1 ΔExon10 cannot( Fig.10 JP and Fig.17 H).

[0140] To investigate how NBR1 participates in autophagy regulation, we first examined the WT and NBR1 ΔExon10 The cellular localization of Fig.17 J). In addition, our studies showed that hnRNPH1 mediates the retention of NBR1 exon 10, which encodes amino acids 288-347 and completely encompasses the CC1 domain (288-329). Therefore, based on the domain structure of NBR1, we constructed NBR1 truncation mutants and performed immunoprecipitation analysis of p62 ( Fig.17 K). Deletion of the CC1 domain significantly impairs the binding of NBR1 to p62 ( Fig.17 L). In-depth analysis revealed that NBR1 can bind to p62, and NBR1 ΔExon10 The binding capacity of Fig.17 M). In addition, NBR1 WT Overexpression leads to a decrease in the number of p62 bodies ( Fig.17 N). These findings suggest that the interaction between NBR1 and p62, mediated by the CC1 domain, is essential for autophagy. Furthermore, the NBR1-p62 interaction maintains cellular homeostasis through autophagy and indirectly inhibits apoptosis.

[0141] In conclusion, our results suggest that the PTK6 / hnRNPH1 axis promotes the inclusion of exon 10 in NBR1, thereby enhancing autophagy and subsequently inhibiting apoptosis.

[0142] Example 8 Tilfrinib inhibits PTK6-activated autophagy, thereby promoting cell apoptosis in vivo and in vitro.

[0143] Tilfrinib is a potent and selective PTK6 inhibitor that exhibits significant antitumor activity, but its effect on CRC has not been reported previously ( Fig.11 A). To address this question, we used Western blot analysis to evaluate the effects of different concentrations of Tilfrinib on the expression of autophagy-related proteins in CRC cells, aiming to determine the optimal treatment concentration. Our results showed that 20 μM Tilfrinib treatment for 24 h was the optimal condition ( Fig.11 B). TEM showed that the number of autophagosomes was significantly reduced under these conditions ( Fig.11 C). Both mCherry-EGFP-LC3 dual fluorescence experiments and Western blot showed that Tilfrinib significantly inhibited PTK6-mediated autophagy ( Fig.11 D, Fig.11 F and Fig.18 A, Fig.18 E). In addition, flow cytometry and Western blot results showed that Tilfrinib significantly attenuated the anti-apoptotic effect mediated by PTK6 ( Fig.11 EF and Fig.18 BE). To further verify its therapeutic potential, we established a CRC patient-derived organoid (PDO) model. The experiment showed that PTK6 significantly promoted PDO formation, while Tilfrinib significantly inhibited PTK6-induced tumor proliferation ( Fig.11 G and Fig.18 F).

[0144] Furthermore, we established a mouse subcutaneous xenograft model and the results showed that Tilfrinib treatment significantly inhibited the increase in tumor volume and weight ( Fig.11 HJ). In addition, Tilfrinib showed significant effects in inhibiting PTK6-induced tumor proliferation ( Fig.11 HJ). IHC analysis of tumors showed that Tilfrinib significantly inhibited PTK6-mediated autophagy ( Fig.11 K and Fig.18 FG). TUNEL assay results showed that the anti-apoptotic effect induced by PTK6 in colorectal cancer cells was effectively reversed by Tilfrinib. Fig.18GH). In addition, we applied a probe targeting NBR1 exon 10 in subcutaneous tumor tissues. The results showed that NBR1 exon 10 skipping events increased significantly after Tilfrinib treatment ( Fig.11 K). At the same time, we detected the LLPS of hnRNPH1 in HCT116 and DLD1 after Tilfrinib treatment and found that the LLPS of hnRNPH1 was also significantly inhibited ( Fig.11 I).

[0145] In conclusion, our experiments demonstrated that Tilfrinib effectively inhibited PTK6-activated autophagy and thus promoted apoptosis both in vivo and in vitro.

[0146] In summary, PTK6 phosphorylates hnRNPH1 at Y210 to promote its phase separation, thereby enhancing the retention of NBR1 exon 10, further promoting autophagy and inhibiting apoptosis in CRC. In addition, we found that Tilfrinib is a potent and selective PTK6 inhibitor that effectively induces apoptosis by targeting autophagy in CRC ( Fig.12 ).

Claims

1. A computer-assisted drug screening method based on PTK6 and hnRNPH1, the method comprising: Obtain PTK6 protein and hnRNPH1 protein data; Selecting the spatial structure of the PTK6 protein and hnRNPH1 protein complex, and determining the complex binding site as the binding site of the targeted drug; A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

2. The computer-assisted drug screening method according to claim 1, wherein the specific steps of the computer-assisted drug screening method are: Obtain the binding site of PTK6 protein and hnRNPH1 protein complex; Based on the spatial structure of the binding site of the PTK6 protein and hnRNPH1 protein complex, compounds with similar structures were screened in the molecular database; The screened compounds were docked with hnRNPH1 protein to calculate the affinity / binding energy of the target receptor to obtain scores, and candidate drugs were obtained by ranking according to the scores; Preferably, the binding site of the PTK6 protein and hnRNPH1 protein complex is amino acid 210 of the hnRNPH1 protein; Preferably, the computer-assisted drug screening method screens protein analogs / antibodies / RNA drugs based on the active sites of the PTK6 protein and hnRNPH1 protein complex; Or screen protein analogs / antibodies / RNA drugs similar to PTK6 protein to obtain candidate drugs; Preferably, the active sites include phosphorylation sites, acetylation sites, and methylation sites.

3. The computer-assisted drug screening method according to claim 1, wherein the specific steps of the computer-assisted drug screening method are: Obtain the binding site of PTK6 protein and hnRNPH1 protein complex; Based on the spatial structure of the binding site of the PTK6 protein and hnRNPH1 protein complex, compounds with similar structures were screened in the molecular database; Perform molecular docking between the screened compounds and PTK6 protein to calculate the affinity / binding energy of the target receptor to obtain a score, and then sort them according to the score to obtain candidate drugs; Preferably, the computer-assisted drug screening method screens protein analogs / antibodies / RNA drugs based on the active sites of the PTK6 protein and hnRNPH1 protein complex; or screens protein analogs / antibodies / RNA drugs similar to the hnRNPH1 protein to obtain candidate drugs; Preferably, the method for determining the binding site of the complex further comprises: Constructing n truncated expression mutants of various PTK6 proteins, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PTK6 and hnRNPH1; Preferably, the key region for the interaction between PTK6 and hnRNPH1 is used as a target site for drug binding; preferably, an immunoprecipitation experiment is performed based on the n truncated expression mutants to obtain the key region for the interaction between PTK6 and the 210th amino acid of the hnRNPH1 protein.

4. A computer-assisted drug screening system based on PTK6 and hnRNPH1, comprising: Data acquisition unit: obtain PTK6 protein and hnRNPH1 protein data; Site determination unit: selecting the spatial structure of the PTK6 protein and hnRNPH1 protein complex, and determining the complex binding site as the binding site of the targeted drug; Drug screening unit: computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

5. A computer-assisted drug screening device based on PTK6 and hnRNPH1, comprising: A processor and a memory, wherein the processor is used to call program instructions, and when the program instructions are called and executed, the computer-assisted drug screening method according to any one of claims 1 to 4 is implemented, and the memory is used to store program instructions.

6. A computer-readable storage medium having a computer program stored thereon, wherein the computer program, when called and executed by a processor, implements the computer-assisted drug screening method according to any one of claims 1 to 4.

7. Applications as described in any of the following: 1) Use of hnRNPH1 inhibitors in the preparation of drugs for treating colorectal cancer; Preferably, the inhibitor of hnRNPH1 reduces the expression level of hnRNPH1, reduces the LLPS of hnRNPH1, reduces the phosphorylation level of hnRNPH1, reduces the acetylation level of hnRNPH1, or reduces the methylation level of hnRNPH1; 2) Application of hnRNPH1 in screening candidate drugs for the treatment of colorectal cancer; 3) Use of an agent that inhibits the binding of PTK6 and hnRNPH1 in the preparation of a drug for treating colorectal cancer; preferably, the agent that inhibits the binding of PTK6 and hnRNPH1 has an action site on the PTK6 protein; Preferably, the agent that inhibits the binding between PTK6 and hnRNPH1 is Tilfrinib; Preferably, the action site of the agent that inhibits the binding of PTK6 and hnRNPH1 is on the hnRNPH1 protein; preferably, the action site of the agent that inhibits the binding of PTK6 and hnRNPH1 is on the 210th amino acid of the hnRNPH1 protein; Preferably, the agent that inhibits the binding between PTK6 and hnRNPH1 acts on the 210th amino acid of the hnRNPH1 protein to reduce its phosphorylation level; 4) Application of the complex formed by the binding of PTK6 and hnRNPH1 in screening drugs for the treatment of colorectal cancer; 5) Application of the complex formed by the binding of PTK6 and hnRNPH1 in screening agents that inhibit the binding of PTK6 and hnRNPH1; 6) Application of PTK6 in preparing regulators of hnRNPH1 phosphorylation levels in cells; 7) Application of PTK6 in preparing cell models of hnRNPH1 hyperphosphorylation / hypophosphorylation; Preferably, the hnRNPH1 hyperphosphorylation / hypophosphorylation is the hyperphosphorylation / hypophosphorylation of the 210th amino acid of the hnRNPH1 protein.

8. A pharmaceutical composition for treating colorectal cancer, comprising an inhibitor of hnRNPH1 and / or an agent that inhibits the binding between PTK6 and hnRNPH1.

9. A method for regulating the phosphorylation level of hnRNPH1 in colorectal cancer cells or tissues in vitro, the method comprising altering the expression of PTK6 in colorectal cancer cells or tissues; Preferably, the method for regulating PTK6 expression in colorectal cancer cells or tissues comprises administering a PTK6 promoter or inhibitor to the colorectal cancer cells or tissues.

10. A method for constructing a colorectal cancer cell model with hyperphosphorylation / hypophosphorylation of hnRNPH1, the method comprising regulating the expression of PTK6 in the cell; Preferably, the method for constructing a colorectal cancer cell model with hyperphosphorylation of hnRNPH1 comprises promoting the expression of PTK6 in the cells; Preferably, the promoting the expression of PTK6 in the cell comprises administering a PTK6 promoter to the cell; Preferably, the method of constructing a colorectal cancer cell model with hypophosphorylation of hnRNPH1 comprises inhibiting the expression of PTK6 in the cells; Preferably, inhibiting the expression of PTK6 in the cell comprises administering a PTK6 inhibitor to the cell; Preferably, the hnRNPH1 hyperphosphorylation / hypophosphorylation is the hyperphosphorylation / hypophosphorylation of the 210th amino acid of the hnRNPH1 protein.

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