Application of CKS1B inhibitors in the treatment of KRAS G12D mutant lung adenocarcinoma

Abstract

This invention discloses the application of CKS1B inhibitors in the treatment of KRAS G12D-mutant lung adenocarcinoma. This invention is the first to discover that inhibiting CKS1B has a good therapeutic effect on KRAS G12D-mutant lung adenocarcinoma. Based on this discovery, this invention also developed a tumor-targeting lipid nanoparticle (siCKS1B@LNP) encapsulating CKS1B siRNA. This nanoparticle can inhibit the proliferation of KRAS G12D-mutant lung adenocarcinoma both in vitro and in vivo, and has high safety. This invention also demonstrates that siCKS1B@LNP does not have a killing effect on lung adenocarcinoma cells of other KRAS mutation types, and has good specificity in the treatment of KRAS G12D-mutant lung adenocarcinoma. This invention provides a novel method for the treatment of KRAS G12D-mutant lung adenocarcinoma and has broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CKS1B inhibitors in the treatment of KRAS G12D mutant lung adenocarcinoma. Background Technology

[0002] Lung cancer is a leading cause of cancer-related death worldwide. Based on histological and cytological characteristics, it can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for a larger proportion and having a poorer prognosis. Early diagnosis and precision treatment of NSCLC are crucial for improving patient outcomes. However, due to the lack of obvious early symptoms, most patients are diagnosed at an advanced stage, missing the optimal treatment window. Furthermore, the high heterogeneity of NSCLC at the genetic and molecular levels leads to significant differences in tumor driver genes, immune microenvironment, and treatment response among patients, making standardized treatment difficult to apply universally.

[0003] Gene mutations are a crucial factor in the development, progression, and metastasis of NSCLC. Key mutated genes influencing NSCLC development include EGFR, ALK, KRAS, BRAF, HER2, ROS1, MET, NTRKs, FGFRs, and NRGs. The KRAS gene is located in the p11.1-p12.1 region of the short arm of chromosome 12, and its genomic structure contains six coding exons spanning approximately 45.5 kb of DNA sequence. The most common KRAS oncogenic mutations are located at codon 12, including G12C, G12D, and G12V. Due to its unique spatial conformation, the KRAS protein lacks typical drug-binding sites, making it a long-standing and difficult-to-target molecular target in drug development. Currently, there are no effective targeted drugs for the KRAS G12D mutation. Therefore, conducting cell biology research related to NSCLC and identifying target molecules for KRAS G12D is of significant scientific importance for the research and treatment of KRAS G12D-related non-small cell lung cancer.

[0004] CKS1B (CDC28 protein kinase regulatory subunit 1B) is a core regulator of cell cycle progression, and its elevated expression is associated with aggressive tumor behavior in various cancer types. However, no studies have yet demonstrated that CKS1B has a targeted therapeutic effect on KRAS G12D-mutant lung adenocarcinoma. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides the application of CKS1B inhibitors in the treatment of KRAS G12D mutant lung adenocarcinoma, providing a safe and effective drug for the treatment of KRAS G12D mutant lung adenocarcinoma, thereby overcoming the current technical problem in the field of the urgent need for effective drugs targeting KRAS G12D mutant lung adenocarcinoma.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: The first aspect of the present invention provides the use of CKS1B inhibitors in the preparation of medicaments for treating KRAS-mutant lung cancer.

[0007] In one embodiment of the invention, treatment means reducing or alleviating, improving or eradicating a disease or one or more symptoms related to the disease. In some embodiments, the term refers to minimizing the spread or worsening of the disease due to the administration of one or more preventive or therapeutic agents to a patient suffering from the disease. For the purposes of the various aspects and embodiments provided by the invention, treatment includes, but is not limited to, reducing, alleviating or improving one or more clinical manifestations or side effects of the treated disease or condition, improving one or more clinical outcomes, reducing the severity of the disease, delaying or slowing the progression of the disease, improving, alleviating or stabilizing the disease state, and other beneficial results described in the invention.

[0008] Furthermore, the CKS1B inhibitors include nucleic acid inhibitors and protein inhibitors.

[0009] Furthermore, the CKS1B inhibitor is a nucleic acid inhibitor.

[0010] Furthermore, the nucleic acid inhibitor includes one or more of siRNA, shRNA, sgRNA, ribozymes, and antisense oligonucleotides.

[0011] Furthermore, the nucleic acid inhibitor is siRNA or sgRNA.

[0012] Furthermore, the siRNA sequence is shown in SEQ ID NO:1-4, and the sgRNA sequence is shown in SEQ ID NO:5.

[0013] In some embodiments, the siRNA refers to a short double-stranded RNA capable of inducing RNA interference by cleaving specific mRNAs. Furthermore, the siRNA is not limited to regions where the double-stranded RNA is perfectly paired, but may include regions where the strands are not paired due to mismatch (corresponding nucleotides are not complementary) or protrusion (absence of nucleotides corresponding to one strand). The siRNA terminal structure may have blunt or protruding ends, as long as the expression of the target gene can be suppressed by the RNA interference effect, and the adhesive end structure may be a 3' protrusion or a 5' protrusion.

[0014] In some embodiments, the shRNA refers to an RNA sequence that produces a strong hairpin bend, which can be used to silence gene expression via RNA interference. Furthermore, the shRNA can be delivered into cells using a vector for cell introduction, and this vector is always delivered to daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure degrades into siRNA via intracellular mechanisms and binds to an RNA-induced silencing complex, which binds to the corresponding mRNA to degrade it.

[0015] In some embodiments, the sgRNA refers to a single-stranded RNA molecule capable of specifically recognizing and binding to the CKS1B gene DNA sequence. Its sequence design is based on the coding region characteristics of the CKS1B gene; for example, a sequence containing specific complementary base arrangements can be used to achieve targeted binding. When the sgRNA binds to the Cas9 nuclease, the resulting ribonucleoprotein complex can recognize a specific region of the CKS1B gene through complementary base pairing. After the endonuclease activity of the Cas9 nuclease is activated, it can cleave the double-stranded DNA of the CKS1B gene under the guidance of the sgRNA, causing gene breakage and triggering the cell's DNA repair mechanism. This gene editing can effectively interfere with the normal expression of the CKS1B gene, thereby inhibiting the synthesis of its encoded protein.

[0016] In some embodiments, the ribozyme refers to an enzyme-RNA molecule capable of catalyzing the cleavage of specific RNA. Specific hybridization of the ribozyme's molecular sequence with complementary target RNA can induce endonuclease cleavage. The ribozyme may include a known sequence responsible for cleaving one or more sequences complementary to or functionally equivalent to the target RNA. Furthermore, the ribozyme may be a hammerhead ribozyme or a Cech-type ribozyme, i.e., a ribonuclease RNA, and may be formed from modified oligonucleotides to improve safety and targeting. Simultaneously, the ribozyme can be distributed in cells expressing the target gene in vivo. DNA constructs encoding the ribozyme under the control of a strong constitutive polymerase III or polymerase II promoter can be used, allowing transfected cells to disrupt endogenous target messengers and produce sufficient amounts of the ribozyme to inhibit translation. Due to its catalytic activity, unlike other antisense molecules, the ribozyme may need to be maintained at low concentrations in the cell.

[0017] In some embodiments, an antisense nucleotide is a sequence that interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to a complementary nucleotide sequence of DNA, immature mRNA, or mature mRNA. Furthermore, because antisense nucleotides are long chains of monomeric units, antisense nucleotides of target RNA sequences can be readily synthesized.

[0018] Furthermore, the KRAS-mutant lung cancer is a KRAS-mutant lung adenocarcinoma.

[0019] Furthermore, the KRAS mutation is a KRAS G12D mutation.

[0020] A second aspect of the present invention provides a medicament for treating KRAS-mutant lung cancer, the medicament comprising a CKS1B inhibitor.

[0021] Furthermore, the CKS1B inhibitors include nucleic acid inhibitors and protein inhibitors.

[0022] Furthermore, the CKS1B inhibitor is a nucleic acid inhibitor.

[0023] Furthermore, the nucleic acid inhibitor includes one or more of siRNA, shRNA, sgRNA, ribozymes, and antisense oligonucleotides.

[0024] Furthermore, the nucleic acid inhibitor is siRNA or sgRNA.

[0025] Furthermore, the siRNA sequence is shown in SEQ ID NO:1-4, and the sgRNA sequence is shown in SEQ ID NO:5.

[0026] Furthermore, the drug is a delivery system that encapsulates a CKS1B nucleic acid inhibitor.

[0027] Furthermore, the delivery system includes plasmids, bacteriophages, virus-derived vectors, or lipid nanoparticles.

[0028] In some embodiments, the vector from which the virus originates is a lentiviral vector, a retroviral vector, adeno-associated virus vector, adenovirus vector, poxvirus vector, herpesvirus vector, or baculovirus vector.

[0029] Furthermore, the delivery system is a lentiviral vector or lipid nanoparticles.

[0030] Furthermore, the lentivirus vector is psPAX2 or PMD2.G.

[0031] Furthermore, the lipid nanoparticles are composed of ionizable lipids, cholesterol, cofactor phospholipids, and polyethylene glycol-modified lipids.

[0032] Furthermore, the lipid nanoparticles are composed of DOTAP, DOPE, cholesterol, and DSPE-PEG.

[0033] Furthermore, the lipid nanoparticles encapsulating the CKS1B nucleic acid inhibitor can be prepared by thin film dispersion, ultrasonic dispersion, reverse phase evaporation, freeze drying, freeze-thaw, double emulsion, or injection.

[0034] In some embodiments, the “thin-film dispersion method” refers to dissolving a membrane material such as phospholipid in an appropriate amount of organic solvent (e.g., chloroform, methanol, or a mixture of chloroform and methanol), then removing the solvent by rotary evaporation under reduced pressure, allowing the lipid to form a thin film on the container wall, adding a buffer solution and shaking, and automatically assembling the lipid film after it detaches, optionally forming liposomes by extrusion.

[0035] In some embodiments, the “ultrasonic dispersion method” refers to a method in which a membrane material such as phospholipids is dissolved in an appropriate amount of chloroform or other organic solvent (optionally, the lipid-soluble drug is dissolved in the organic solvent), and then the solvent is removed by rotary evaporation under reduced pressure, so that the lipids form a thin film on the container wall. After that, a buffer solution is added, and the lipid film is detached and formed into liposomes by ultrasonic treatment.

[0036] In some embodiments, the "reverse phase evaporation method" is also known as the reverse phase evaporation method, which refers to dissolving phospholipids or other membrane materials in an appropriate amount of chloroform or other organic solvents, optionally adding an aqueous solution of the drug to be encapsulated, performing short-term sonication to form a stable water-in-oil (W / O) emulsion, then removing the organic solvent by vacuum evaporation, reaching a colloidal state, adding buffer solution, rotating to detach the gel from the container wall, obtaining an aqueous suspension, and then optionally removing the unencapsulated drug by gel chromatography or ultracentrifugation to obtain liposomes (preferably large single-compartment liposomes).

[0037] In some embodiments, the "freeze-drying method" refers to highly dispersing a membrane material such as phospholipids in an aqueous solution, freeze-drying it, and then dispersing it in an aqueous medium (optionally containing a drug) to form liposomes.

[0038] In some implementations, the "freeze-thaw method" refers to dissolving lipid materials such as phospholipids in a small amount of water, melting cholesterol and mixing it with the solution, and then dripping the mixture into an aqueous solution at about 65°C and keeping it warm to obtain liposomes.

[0039] In some implementations, the "double emulsion method" refers to emulsifying a small amount of aqueous phase with a larger amount of phospholipid oil phase to form W / O type reverse micelles. After removing part of the solvent by vacuum evaporation, a larger amount of aqueous phase is added for a second emulsification to form a water-in-oil-in-water (W / O / W) type double emulsion. After removing the organic solvent by vacuum evaporation, liposomes are obtained.

[0040] In some implementations, the "injection method" refers to dissolving phospholipids or other lipid materials in an appropriate amount of organic solvent to form an oil phase, then injecting the oil phase at a uniform rate into a constant-temperature aqueous phase above the boiling point of the organic solvent, stirring to allow the organic solvent to completely evaporate, and then homogenizing or sonicating to obtain liposomes.

[0041] Furthermore, the lipid nanoparticles encapsulating the CKS1B nucleic acid inhibitor were prepared by an ethanol injection method.

[0042] Furthermore, the lipid nanoparticles encapsulating the CKS1B nucleic acid inhibitor have a particle size of 89.17±3.41 nm and a zeta potential of 21.16±1.99.

[0043] In this invention, the term "zeta potential" is a quantity related to the surface charge of particles in a liquid, indicating the potential stability of a solid dispersed in a liquid or a liquid dispersed in a liquid. If all particles in a suspension have a large negative or positive zeta potential, they tend to repel each other and have no tendency to flocculate. However, if particles have a low zeta potential value, there will be a smaller force preventing particle aggregation, and the particles may have a greater tendency to flocculate. More information on zeta potential can be found in Hunter, RJ (1988), Zeta Potential in Colloid Science: Principles and Applications, Academic Press, UK.

[0044] The CKS1B nucleic acid inhibitor delivery system of the present invention can significantly inhibit CKS1B expression intracellularly. In some embodiments, CKS1B expression is inhibited by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and even 100%. Further, the CKS1B nucleic acid inhibitor delivery system of the present invention can inhibit CKS1B expression by at least 50%. Even further, the CKS1B nucleic acid inhibitor delivery system of the present invention can inhibit CKS1B expression by at least 70%.

[0045] The third aspect of the present invention provides any of the following products: 1) A pharmaceutical composition for treating KRAS-mutant lung cancer, said pharmaceutical composition comprising the drug described in the second aspect of the present invention and other drugs that can be used to treat KRAS-mutant lung cancer.

[0046] 2) A pharmaceutical preparation for treating KRAS-mutant lung cancer, said pharmaceutical preparation comprising the medicament described in the second aspect of the present invention and pharmaceutically acceptable excipients.

[0047] 3) A derivative of a CKS1B inhibitor, said derivative comprising a chemically modified CKS1B inhibitor.

[0048] In some implementations, the other drugs that can be used to treat KRAS-mutant lung cancer include KRAS inhibitors, immune checkpoint inhibitors (such as pembrolizumab, nivolumab, and atezolizumab), and chemotherapy drugs (such as paclitaxel in combination with cisplatin (TP) or pemetrexed).

[0049] In some embodiments, the other drugs that can be used to treat KRAS-mutant lung cancer are not limited to the specific drugs listed above in this invention, and any drug that may be used to treat KRAS-mutant lung cancer will fall within the protection scope of this invention.

[0050] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used non-toxic, pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to patients / subjects via any route, provided that the target tissue can be reached.

[0051] In some implementations, the patient / subject can be human or non-human and can include, for example, animal strains or species used as a "model system" for research purposes. Similarly, the patient / subject can include adults or adolescents (e.g., children). Furthermore, the patient / subject can refer to any living organism, preferably a mammal (e.g., human or non-human). Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (e.g., chimpanzees) and other apes and monkeys; livestock such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0052] In some embodiments, the two drugs in the pharmaceutical composition can be administered simultaneously, separately, or sequentially. Simultaneous administration means that the two drugs are administered concurrently. If not administered simultaneously, they are administered sequentially within a time frame so that both can be therapeutically effective within the same time frame. Therefore, sequential administration allows for the administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for a simultaneously therapeutically effective amount of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives. In contrast to simultaneous or sequential administration, the interval between administration of one drug and another is significant, meaning that when the second drug is administered, the first administered drug may no longer be present in the bloodstream at a therapeutically effective amount.

[0053] In some embodiments, the therapeutically effective amount refers to the amount of an active compound or pharmaceutical agent that elicits a biological or medical response sought by researchers, veterinarians, physicians, or other clinicians in a tissue, system, animal, or human. The therapeutically or pharmaceutically effective amount of the compound to be administered will be determined by such considerations and is the minimum amount necessary to improve, cure, or treat a disease or condition or one or more symptoms thereof. The pharmaceutical composition of the invention will be formulated, administered, and applied in a manner consistent with good medical practice, i.e., the dosage, concentration, regimen, process, medium, and route of administration. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery, the method of administration, the administration regimen, and other factors known to a medical practitioner, such as the individual patient's age, weight, and response.

[0054] In some embodiments, specific examples of the pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl chloride; benzalkonium chloride, benzyl chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); proteins such as serum albumin, gelatin, or immunoglobulins; and hydrophilic substances. Polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEENTM, PLURONICS™, or polyethylene glycol (PEG).

[0055] Furthermore, the chemical modifications include ribose modification, base modification, phosphate ester skeleton modification, and terminal modification.

[0056] Furthermore, the terminal modifications include cholesterol modification, PEG modification, labeling modification, photosensitization modification, pH-responsive modification, and targeted ligand modification.

[0057] Furthermore, the labeling modification involves directly or indirectly coupling the CKS1B inhibitor to a detectable label.

[0058] Furthermore, the detectable markers include fluorescent dyes, enzymes, chemiluminescent markers, radioactive isotopes, electron-dense reagents, colored particles, biotin, or digoxin.

[0059] In some implementations, the detectable marker often generates a measurable signal, such as radioactivity, fluorescence, color, or enzyme activity. Examples of suitable fluorescent dyes include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, Cy5 / Cy5.5, dansyl chloride, and phycoerythrin; examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; chemiluminescent markers include, but are not limited to, luminol and its derivatives, isoluminol and its derivatives, acridine esters and their derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes; radioactive isotopes include, but are not limited to, those that... 68 Ga、 86 Y、 110 In、 111 In、 177 Lu、 18F, 52 Fe、 62 Cu、 64 Cu、 11 C 67 Cu、 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 15 O. The aforementioned detectable markers can be directly connected to or coupled to the drug or indirectly through an intermediate such as a connector known in the art, using techniques known in the art.

[0060] Furthermore, the detectable marker is Cy5.5.

[0061] The fourth aspect of the present invention provides for any of the following applications: 1) Application of CKS1B inhibitors in the preparation of pharmaceutical compositions for treating KRAS-mutant lung cancer.

[0062] 2) Application of CKS1B inhibitors in the preparation of drug formulations for the treatment of KRAS-mutant lung cancer.

[0063] 3) Application of CKS1B inhibitors in the preparation of CKS1B inhibitor derivatives.

[0064] The fifth aspect of the present invention provides a method for inhibiting the proliferation of KRAS-mutant lung cancer cells in vitro, the method comprising treating a system containing KRAS-mutant lung cancer cells with the drug described in the second aspect of the present invention or the product described in the third aspect of the present invention.

[0065] Furthermore, the system includes a cellular system, a subcellular system, a tissue system, or an organ system.

[0066] Furthermore, the method described is not for therapeutic purposes.

[0067] Advantages and beneficial effects of the present invention: This invention is the first to discover that inhibiting CKS1B has a good therapeutic effect on KRAS G12D mutant lung adenocarcinoma. Based on this discovery, this invention also developed a tumor-targeting lipid nanoparticle (siCKS1B@LNP) encapsulating CKS1B siRNA. This nanoparticle can inhibit the proliferation of KRAS G12D mutant lung adenocarcinoma both in vitro and in vivo, and has high safety. This invention also demonstrates that siCKS1B@LNP has no killing effect on lung adenocarcinoma cells of other KRAS mutation types, and has good specificity in the treatment of KRAS G12D mutant lung adenocarcinoma. This invention provides a novel method for the treatment of KRAS G12D mutant lung adenocarcinoma and has broad clinical application prospects. Attached Figure Description

[0068] Figure 1 Figure 1 shows the effect of altered CKS1B gene expression on KRAS G12D mutant lung adenocarcinoma mice. In the figure, A is a schematic diagram of the experimental procedure, B is the lung tissue section and tumor burden quantitative analysis of KRAS G12D mutant lung adenocarcinoma mice after CKS1B overexpression, C is the IHC verification of the CKS1B overexpression effect, D is the lung tissue section and tumor burden quantitative analysis of KRAS G12D mutant lung adenocarcinoma mice after CKS1B knockout, and E is the IHC verification of the CKS1B knockout effect.

[0069] Figure 2 Figure 1 shows the physicochemical properties of siCKS1B@LNP. In the figure, A is a schematic diagram of the synthesis process of siCKS1B@LNP, B is the morphology of siCKS1B@LNP under transmission electron microscopy, C is the hydrodynamic particle size distribution of siCKS1B@LNP, D is the Zeta potential of siCKS1B@LNP, E is the colloidal stability of siCKS1B@LNP, F is the endocytosis of siCKS1B@LNP in A549 cells, G is the quantification of F, H is the time dependence of siCKS1B@LNP uptake and intracellular transport, I is the efficacy of siCKS1B@LNP in silencing CKS1B, and J is the cytotoxicity of siCKS1B@LNP.

[0070] Figure 3 The in vivo therapeutic effects of siCKS1B@LNP are shown in Figure A, where A represents lung tissue sections and quantitative analysis of tumor burden in mice with KRAS G12D mutant lung adenocarcinoma after siCKS1B@LNP treatment, and B represents the change in body weight of the mice.

[0071] Figure 4 The in vitro targeting effect of siCKS1B@LNP is shown in Figure B. A represents the cck8 results of different mutant Kras cell lines treated with siCKS1B@LNP, B represents the plate clone results of different mutant Kras cell lines treated with siCKS1B@LNP, and C represents the quantification of Figure B. Detailed Implementation

[0072] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0073] Table 1. Sequence information used in this invention

[0074] Example 1: Study on the effect of CKS1B gene on KRAS G12D mutant lung adenocarcinoma I. Experimental Methods 1. The plasmid Plvx-tetOn-mouse-CKS1B-3×Flag was constructed using conventional methods, with the GeneID of CKS1B being 1163; the plasmid pSECC-CKS1B-sgRNA was constructed using conventional methods, and the CKS1B-sgRNA sequence is shown in Table 1.

[0075] Lentiviral construction: The above plasmids, along with the lentiviral packaging plasmid psPAX2 and the lentiviral envelope plasmid pMD2.G (Addgene product numbers #12260 and #12259), were co-transfected into HEK 293T cells using VigoFect transfection reagent (Vigorous Biotechnology). The culture medium was changed 6 hours after transfection, and the virus-containing supernatant was collected 48 hours later. After filtration through a 0.45 μm filter, the supernatant was used directly for in vitro stable cell line construction. For in vivo delivery, the lentivirus was concentrated by ultracentrifugation (27,000 rpm, 4°C, 2 hours) and then resuspended overnight in Opti-MEM medium before administration.

[0076] 2. Animals: All animal experiments were approved by the Institutional Animal Care and Use Committee of Tangdu Hospital, Fourth Military Medical University. Mice were randomly assigned to experimental groups.

[0077] In the overexpression experiment, Teto-KrasG12D / CC10rtTA mice (constructed and kindly provided by Professor Chen Liang's research group at the School of Life Science and Technology, Jinan University) were intranasally inoculated with lentivirus (Plvx-tetOn-mouse-CKS1B-3×Flag or control group) and fed doxycycline (Dox) diet for 2 months to induce tumor development. Subsequently, lung tissue was collected for hematoxylin-eosin (H&E) staining and immunohistochemistry (antibody CKSIB: Abways, AY1698).

[0078] In the knockout experiment, 6-week-old female LSL-KrasG12D mice (Nanmo Biotechnology, NM-KI-190003) (n=5) were treated with CKS1B-targeting sgRNA lentivirus via intranasal administration for 10 weeks. Subsequently, lung tissue was collected for hematoxylin-eosin (H&E) staining and immunohistochemistry (antibody CKSIB: Abways, AY1698).

[0079] The construction principle of the Teto-KrasG12D / CC10rtTA mouse, and the specific construction method, can be found in (Zhang H, Cao J, Li L et al. Identification of urine protein biomarkers with the potential for early detection of lung cancer. Sci Rep. 2015 Jul 2;5:11805.): Teto-KrasG12D mice need to be bred with CC10rtTA mice to achieve a double positive result in order to function effectively. The core of the CC10rtTA mouse lies in linking the coding sequence of the Clara cell-specific promoter (CC10) in the lungs with that of tetracycline transactivator (rtTA). The CC10rtTA fusion gene is transferred into mouse zygotes via pronuclear microinjection, allowing it to integrate into the genome, thereby obtaining transgenic mice that specifically express rtTA protein in lung epithelial cells. rtTA cannot function without Dox; once the mice are fed Dox, rtTA is activated, thereby initiating the expression of downstream genes. The core of the Teto-KrasG12D mouse is the linking of a tetracycline-responsive element (TetO) that responds to the rtTA protein to a mutated Kras gene (KrasG12D). This structure is randomly inserted into the mouse genome via microinjection to obtain Teto-KrasG12D transgenic mice. When Teto-KrasG12D mice are mated with CC10rtTA mice, their offspring carry both components. Only in these double-positive offspring can oral administration of Dox activate lung-specific rtTA expression, thereby initiating the expression of the KrasG12D mutated gene and ultimately inducing lung adenocarcinoma.

[0080] II. Experimental Results We proceeded according to the established plan ( Figure 1 A), in the Teto-KrasG12D / CC10rtTA model, CKS1B overexpression was achieved via intranasal lentivirus delivery. Figure 1 C), in the LSL-KrasG12D model, CKS1B gene knockout was achieved via intranasal lentiviral delivery. Figure 1 E). Notably, CKS1B overexpression accelerated tumor progression in KrasG12D-driven lung adenocarcinoma (LUAD). Figure 1 B), while CKS1B gene knockout significantly inhibited tumor growth in the same genetic background ( Figure 1D). These contrasting phenotypic effects clearly demonstrate that CKS1B does indeed play a carcinogenic driver role in the development of LUAD with KrasG12D mutations, and that inhibiting CKS1B can effectively treat LUAD with KrasG12D mutations.

[0081] Example 2: Preparation and in vivo efficacy verification of lipid nanoparticles encapsulating CKS1B siRNA (siCKS1B@LNP) I. Experimental Methods 1. Preparation method of siCKS1B@LNP: siCKS1B@LNP was synthesized via a modified ethanol injection method. 3.5 mg DOTAP, 0.5 mg DOPE, 0.5 mg cholesterol, and 0.5 mg DSPE-PEG were dissolved in 2 mL of anhydrous ethanol and gently stirred at 37 °C. siCKS1B (sequence shown in Table 1) was dissolved in 50 mM citrate buffer (pH 4.0) containing 25% (v / v) ethanol, at a concentration of 0.08 mg / mL. The siRNA solution was slowly added dropwise to the lipid solution under magnetic stirring, followed by gentle mixing for 20 minutes to allow the lipid nanoparticles to self-assemble at room temperature. The resulting mixture was sonicated using a probe sonicator (JP-020, 180 W, 10 min) to reduce particle size and improve uniformity. Subsequently, the sonicated suspension was extruded 20 times through a liposome extruder and passed through a 100 nm filter membrane to obtain nanoparticles with uniform and controllable particle size. To remove unencapsulated siRNA and ethanol, the prepared siCKS1B@LNP was transferred to a dialysis bag (30 nm pore size polycarbonate membrane) and dialyzed with PBS buffer (pH 7.4) at 4 °C for 24 hours, with the buffer changed three times during this period. The final siCKS1B@LNP suspension was collected, filtered through a 0.22 μm syringe filter, and stored at 4 °C.

[0082] 2. Characterization of siCKS1B@LNP: The particle size and zeta potential of siCKS1B@LNP were determined using a NanoBrook90plusPALS nanoparticle size and zeta potential analyzer, and its morphology was observed using a transmission electron microscope (TF20). To assess stability, siCKS1B@LNP was stored at 4°C, and particle size was measured on day 1 and day 7. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency (%) = (dialyzed siRNA - undialyzed siRNA) / (dialyzed siRNA) × 100 3. Subcellular localization of Cy5.5-siCKS1B@LNP: A549 cells (Chinese Academy of Sciences Cell Bank, catalog number: SCSP-503) were counted and seeded in 6 cm culture dishes. The next day, after transfection with Cy5.5-labeled siCKS1B@LNP, the cells were incubated at 37°C for 6 or 12 hours. Then, pre-warmed (37°C) lysosomal tracking agent green dye solution (diluted 1:10,000) was added, and incubation continued for 30 minutes. After discarding the culture medium, the cells were washed three times with PBS. Then, 1 mL of 10 μg / mL Hoechst 33342 was added for staining for 10 minutes. Finally, representative fields of view were imaged using a confocal microscope (Olympus FV3000).

[0083] 4. Real-time quantitative PCR Total RNA was extracted using Trizol reagent (Invitrogen), and qPCR reverse transcription was performed using SynScript III RT SuperMix (Qingdao Science & Technology Co., Ltd.). qPCR was then performed on a CFX96 deep-well real-time system (Bio-Rad) using ArtiCanCEO SYBR qPCRMix (Qingdao Science & Technology Co., Ltd.). Gene expression was normalized using β-actin as an internal control and analyzed by 2^( The analysis was performed using the ΔΔCt method. Primer sequences are shown in Table 1.

[0084] 5. Animals: Teto-KrasG12D / CC10rtTA mice were divided into groups and treated with siCKS1B@LNP or siNC@LNP 40μl via intranasal route. Lung tissue was then collected for hematoxylin-eosin (H&E) staining.

[0085] II. Experimental Results Physicochemical characterization showed that siCKS1B@LNP formed a monodisperse spherical nanostructure. Figure 2 B), with a particle size of 89.17±3.41 nm, a low polydispersity index (PDI=0.226±0.013), and a zeta potential of 21.16±1.99 ( Figure 2 C, 2D). This formulation exhibits excellent colloidal stability, maintaining its size distribution and showing no significant aggregation after 7 days of storage at 4°C. Figure 2 E). Cellular internalization studies using Cy5.5-labeled nanoparticles showed time-dependent accumulation in A549 cells and significant co-localization in the late endosome compartment, confirming efficient endocytic uptake. Figure 2 F-2H). Functional validation showed that siCKS1B@LNP treatment achieved potent CKS1B gene silencing (>70% reduction). Figure 2I), and did not induce cytotoxic effects ( Figure 2 J).

[0086] In a KrasG12D transgenic lung adenocarcinoma mouse model, intranasal administration of siCKS1B@LNP resulted in a significant reduction in tumor burden of nearly 80%. Histopathological analysis (H&E staining) confirmed a significant reduction in tumor lesions, which was associated with a significant treatment response. Figure 3 A). Furthermore, this potent antitumor effect did not cause systemic toxicity, body weight remained stable during treatment, and no histopathological abnormalities were observed in major organs. Figure 3 B, Table 2).

[0087] Table 2 Serum Biochemical Indicators

[0088] Example 3: In vitro validation of siCKS1B@LNP targeting KRAS G12D mutant lung adenocarcinoma I. Experimental Methods Stable cell lines of common clinical Kras mutants were constructed using H1299 cells (Chinese Academy of Sciences Cell Bank, catalog number: SCSP-589), including three common mutations: KRAS G12D, KrasG12C, and KrasG12V. Different Kras mutant plasmids were constructed using the PCDH-EF1A plasmid combined with homologous recombination (Tsingke Biotechnology Co., Ltd). Then, using VigoFect transfection reagent (Vigorous Biotechnology), the lentiviral plasmids PCDH-EF1A-KRAS G12D / KrasG12C / KrasG12V were co-transfected into HEK293T cells with packaging plasmids psPAX2 and pMD2.G (Addgene #12260 and #12259), respectively. The culture medium was changed 6 hours after transfection. After 48 hours, the virus-containing supernatant was collected, filtered through a 0.45 μm filter membrane, and co-cultured directly with H1299 cells for 8 hours. Then, the culture medium was replaced with fresh medium and cultured for another 24 hours. Stable transduced cells were then screened with 1 μg / mL puromycin for 72 hours to obtain stable H1299 cell lines with different Kras mutations.

[0089] siCKS1B@LNP was transfected into H1299 stable cell lines with different Kras mutations. The cells were then cultured at 37°C for subsequent CCK8 assays and plate colony experiments.

[0090] II. Experimental Results To verify the specificity of siCKS1B@LNP in treating KRAS G12D mutant lung adenocarcinoma, we first constructed stable H1299 cell lines expressing different KRAS mutants (KRAS G12D, KRAS G12C, and KRAS G12V). Stable cell lines expressing these three KRAS mutants were successfully obtained through lentiviral infection and puromycin selection. Subsequently, we evaluated the effect of siCKS1B@LNP on the proliferation of these different KRAS mutant cells. As shown in the figure, with the untreated control group's cell viability set at 100% as a reference, the survival rate of KRAS G12D mutant cells was significantly reduced after siCKS1B@LNP treatment, indicating that siCKS1B@LNP had the most significant inhibitory effect on the KRAS G12D mutation. In contrast, the survival rates of KRAS G12C and KRAS G12V mutant cells showed no significant difference compared to the control group. These results indicate that siCKS1B@LNP can specifically inhibit the proliferation of KRAS G12D mutant lung adenocarcinoma cells. Figure 4 This suggests that the nanotherapy strategy has specific therapeutic potential against the KRAS G12D mutation subtype.

[0091] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of CKS1B inhibitors in the preparation of drugs for treating KRAS-mutant lung cancer.

2. The application according to claim 1, characterized in that, The CKS1B inhibitors include nucleic acid inhibitors and protein inhibitors; Preferably, the CKS1B inhibitor is a nucleic acid inhibitor; Preferably, the nucleic acid inhibitor includes one or more of siRNA, shRNA, sgRNA, ribozymes, and antisense oligonucleotides; Preferably, the nucleic acid inhibitor is siRNA or sgRNA; Preferably, the siRNA sequence is shown in SEQ ID NO:1-4, and the sgRNA sequence is shown in SEQ ID NO:

5.

3. The application according to claim 1, characterized in that, The KRAS-mutant lung cancer is a KRAS-mutant lung adenocarcinoma; Preferably, the KRAS mutation is a KRAS G12D mutation.

4. A drug for treating KRAS-mutant lung cancer, characterized in that, The drugs include CKS1B inhibitors; Preferably, the CKS1B inhibitor includes a nucleic acid inhibitor and a protein inhibitor; Preferably, the CKS1B inhibitor is a nucleic acid inhibitor; Preferably, the nucleic acid inhibitor includes one or more of siRNA, shRNA, sgRNA, ribozymes, and antisense oligonucleotides; Preferably, the nucleic acid inhibitor is siRNA or sgRNA; Preferably, the siRNA sequence is shown in SEQ ID NO:1-4, and the sgRNA sequence is shown in SEQ ID NO:

5.

5. The drug according to claim 4, characterized in that, The drug is a delivery system that encapsulates a CKS1B nucleic acid inhibitor; Preferably, the delivery system comprises plasmids, bacteriophages, virus-derived vectors, or lipid nanoparticles; Preferably, the delivery system is a lentiviral vector or lipid nanoparticles; Preferably, the lentiviral vector is psPAX2 or PMD2.G; Preferably, the lipid nanoparticles are composed of ionizable lipids, cholesterol, cofactor phospholipids, and polyethylene glycol-modified lipids; Preferably, the lipid nanoparticles are composed of DOTAP, DOPE, cholesterol, and DSPE-PEG.

6. The drug according to claim 5, characterized in that, The lipid nanoparticles can be prepared by thin film dispersion, ultrasonic dispersion, reverse phase evaporation, freeze drying, freeze-thaw, double emulsion, or injection. Preferably, the lipid nanoparticles are prepared by an ethanol injection method; Preferably, the lipid nanoparticles have a particle size of 89.17±3.41 nm and a zeta potential of 21.16±1.

99.

7. Any of the following products: 1) A pharmaceutical composition for treating KRAS-mutant lung cancer, said pharmaceutical composition comprising the drug as described in any one of claims 4-6 and other drugs that can be used to treat KRAS-mutant lung cancer; 2) A pharmaceutical preparation for treating KRAS-mutant lung cancer, said pharmaceutical preparation comprising the drug as described in any one of claims 4-6 and pharmaceutically acceptable excipients; 3) A derivative of a CKS1B inhibitor, said derivative comprising a chemically modified CKS1B inhibitor.

8. The product according to claim 7, characterized in that, The chemical modifications include ribose modification, base modification, phosphate ester skeleton modification, and terminal modification; Preferably, the terminal modification includes cholesterol modification, PEG modification, labeling modification, photosensitization modification, pH-responsive modification, and targeted ligand modification; Preferably, the labeling modification involves directly or indirectly coupling the CKS1B inhibitor to a detectable label; Preferably, the detectable markers include fluorescent dyes, enzymes, chemiluminescent markers, radioactive isotopes, electron-dense reagents, colored particles, biotin, or digoxin; Preferably, the detectable marker is Cy5.

5.

9. Any of the following applications: 1) Application of CKS1B inhibitors in the preparation of pharmaceutical compositions for treating KRAS-mutant lung cancer; 2) Application of CKS1B inhibitors in the preparation of drug formulations for the treatment of KRAS-mutant lung cancer; 3) Application of CKS1B inhibitors in the preparation of CKS1B inhibitor derivatives.

10. A method for inhibiting the proliferation of KRAS-mutant lung cancer cells in vitro, characterized in that, The method includes treating a system containing KRAS-mutant lung cancer cells with the drug as described in any one of claims 4-6 or the product as described in claim 7 or 8. Preferably, the system includes a cellular system, a subcellular system, a tissue system, or an organ system; Preferably, the method is a non-therapeutic method.

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