Application of HS2ST1 inhibitor in preparation of medicine for treating KRAS inhibitor drug-resistant tumors

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an HS2ST1 inhibitor in preparation of a medicine for treating KRAS inhibitor drug-resistant tumors. At present, more than 50% of KRAS inhibitor drug-resistant patients have unclear mechanisms and lack of effective intervention targets, and HS2ST1 is identified as a key regulation target of KRAS inhibitor drug resistance for the first time. Researches find that the expression of HS2ST1 in KRAS inhibitor drug-resistant cells is significantly up-regulated; functional experiments prove that targeted intervention of the HS2ST1 can effectively enhance the curative effect of the KRAS inhibitor and reverse the drug resistance phenotype of the KRAS inhibitor, and a new strategy and a potential treatment direction are provided for overcoming KRAS targeted treatment drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of HS2ST1 inhibitors in the preparation of drugs for treating KRAS inhibitor-resistant tumors. Background Technology

[0002] The Ras oncogene family includes KRAS, HRAS, and NRAS. KRAS mutations are particularly common, frequently leading to fatal malignant tumors such as pancreatic cancer (87%), colorectal cancer (43%), and lung adenocarcinoma (33%), and are significantly associated with poor patient prognosis. KRAS mutations commonly occur at the 12th codon, with G12D mutations being the most frequent in pancreatic and urea-cell cancers (49% and 44%, respectively); and G12C mutations (44%) being the most frequent in lung adenocarcinomas. KRAS proteins function as key signaling proteins within cells, acting as "molecular switches." Their activity is regulated by guanine nucleotide binding status. Under the influence of guanine nucleotide exchange factors, KRAS binds to GTP and becomes activated, initiating downstream signaling pathways (such as RAF-MEK-ERK, PI3K-AKT-mTOR, etc.) to promote cell proliferation and differentiation. Conversely, when KRAS binds to GTPase activators, it catalyzes the hydrolysis of GTP to GDP, returning KRAS to its inactive state. Under normal physiological conditions, KRAS is rapidly inactivated after activation, and signal transduction is transient. Mutations in the KRAS gene (such as G12C / D) disrupt its interaction with GTPase-activating proteins, significantly reducing GTP hydrolysis efficiency, leading to KRAS being persistently locked in a GTP-bound activated state. This abnormal activation causes downstream target signaling pathways to become persistently overactive, driving uncontrolled cell proliferation, differentiation disorders, and tumor formation.

[0003] Currently, the benefits of targeted therapy for KRAS-mutant tumors remain very limited. Because the KRAS protein surface is smooth and lacks binding pockets, it is difficult to directly inhibit, and there is still a lack of widely applicable drugs targeting this target in clinical practice. A 2013 study discovered that when the KRAS gene undergoes a G12C mutation, the glycine encoded by codon 12 is replaced by cysteine, leading to a significant increase in the electrophilicity of the thiol group of the cysteine ​​residue, exposing a novel allosteric binding pocket located in the Switch II pocket. This discovery overturned the traditional perception that KRAS is "undrugable" and laid the structural foundation for targeted therapy. Over the past decade, two KRAS G12C covalent inhibitors developed based on this binding pocket have been launched. However, Amgen's sotorasib was withdrawn from the market in December 2023 due to reliability issues with data from its Phase III clinical trial (CodeBreaK 200). Currently, the only commercially available KRAS inhibitor globally is adagrasib. Its mechanism of action involves forming an irreversible covalent bond with the cysteine ​​residue at position 12 of the KRAS G12C mutant protein, permanently locking it in an inactive GDP-binding state. Besides adagrasib, other drugs targeting the KRAS G12D mutation include MRTX-1133, RMC-9805, and HRS-4642, as well as the pan-KRAS inhibitor RMC-6236, but these are still in clinical trials.

[0004] HS2ST1 (heparan sulfate 2-O-sulfotransferase 1) is a key enzyme in the heparan sulfate biosynthetic pathway. Its core mechanism of action involves using the sulfate donor 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) to transfer a sulfonic acid group (SO3) to the sulfonic acid group. 2- HS2ST1 specifically translocates to the C2 site of hexuronic acid residues in the heparan sulfate chain, thereby precisely regulating the sulfation modification pattern of heparan sulfate. This precise modification is crucial for the function of heparan sulfate, as it is widely involved in cell signaling, cell adhesion, growth factor binding, and various biological processes. Although previous studies have suggested a possible association between abnormal HS2ST1 expression and the development of malignant tumors such as prostate cancer, leukemia, breast cancer, and head and neck squamous cell carcinoma, its role as a "cancer promoter" or "cancer suppressor" in these cancers remains unclear. Currently, research on the biological function of HS2ST1 and its interaction with oncogenic KRAS signaling pathways has not been reported in tumor types with high KRAS mutation rates (such as pancreatic cancer, colorectal cancer, and lung adenocarcinoma).

[0005] As the only marketed KRAS inhibitor, adagrasib showed good efficacy in the early stages of treatment, but resistance soon emerged. Studies have shown that in patients with advanced KRAS G12C-mutant non-small cell lung cancer (NSCLC), the objective response rate was 42.9%, the median overall survival was 12.6 months, and the median progression-free survival was only 6.5 months. Studies have shown that only 45% of adagrasib-resistant patients had detectable resistance targets. These mechanisms include (1) KRAS-dependent resistance: secondary KRAS mutations / KRAS gene amplification; (2) non-KRAS-dependent resistance: ① abnormal activation of upstream RTK (such as EGFR mutations; MET amplification) leading to reactivation of the RAS / MAPK and PI3K / AKT pathways; ② downstream effector molecule mutations: such as activating mutations like NRAS (Q61K), BRAF (V600E), and PIK3CA amplification, which directly activate the MAPK and PI3K / AKT pathways; ③ histological transformation. Therefore, finding new drug resistance targets is an urgent task to be solved in addressing the problem of adagrasib resistance. Summary of the Invention

[0006] To address the aforementioned technical issues, this invention aims to explore the role of HS2ST1 in patients resistant to KRAS inhibitors and investigate whether it can become a new target for reversing drug resistance.

[0007] The first aspect of the present invention aims to provide the use of HS2ST1 inhibitors in the preparation of medicaments for treating KRAS inhibitor-resistant tumors.

[0008] The second aspect of this invention aims to provide the application of the combination of HS2ST1 inhibitors and KRAS inhibitors in the preparation of medicaments for treating tumors.

[0009] A third aspect of the present invention is to provide a pharmaceutical composition.

[0010] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the invention provides the use of an HS2ST1 inhibitor in the preparation of a medicament for treating KRAS inhibitor-resistant tumors.

[0011] In some embodiments of the present invention, the HS2ST1 inhibitor comprises at least one of the following: (a1) Substances that inhibit the activity of HS2ST1 protein; (a2) Substances that reduce the content of HS2ST1 protein; (a3) Substances that knock out the HS2ST1 gene; (a4) Substances that inhibit the expression of the HS2ST1 gene.

[0012] Among them, the CDS sequence of the HS2ST1 gene is (5 -3 ）：

[0013] The amino acid sequence of HS2ST1 (N-terminus to C-terminus) is as follows: MGLLRIMMPPKLQLLAVVAFAVAMLFLENQIQKLEESRSKLERAIARHEVREIEQRHTMDGPRQDATLDEEEDMVIIYNRVPKTASTSFTNIAYDLCAKNKYHVLHINTTKNNPVMSLQDQVRFVKNITSWKEMKPGFYHGHVSYLDFAKFGVKKKPIYINVIRDPIERLVSYYYFLRFG DDYRPGLRRRKQGDKKTFDECVAEGGSDCAPEKLWLQIPFFCGHSSECWNVGSRWAMDQAKYNLINEYFLVGVTEELEDFIMLLEAALPRFFRGATELYRTGKKSHLRKTTEKKLPTKQTIAKLQQSDIWKMENEFYEFALEQFQFIRAHAVREKDGDLYILAQNFFYEKIYPKSN (SEQ ID NO: 2).

[0014] In some embodiments of the present invention, the HS2ST1 inhibitor includes nucleic acid molecules, protein molecules, and small molecule compounds.

[0015] In some embodiments of the present invention, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide.

[0016] In some embodiments of the present invention, the sequence of the shRNA is as shown in SEQ ID NO: 7 or 8.

[0017] In some embodiments of the present invention, the protein molecule is a specific antibody against HS2ST1.

[0018] In some embodiments of the present invention, the small molecule compound is a small molecule compound that inhibits HS2ST1.

[0019] In some embodiments of the present invention, the KRAS inhibitor includes an inhibitor targeting KRAS gene mutations or a broad-spectrum KRAS inhibitor.

[0020] In some embodiments of the present invention, the broad-spectrum KRAS inhibitors include: RMC-6236, PF-07985045, LY4066434, QTX3544, and QTX3034.

[0021] In some embodiments of the present invention, the KRAS gene mutations include KRAS G12C, KRAS G12D, and KRASG12V.

[0022] In some embodiments of the present invention, the inhibitors against KRAS G12C gene mutations include: Sotorasib, Adagrasib, Fulzerasib, Garsorasib, and Glecirasib.

[0023] In some embodiments of the present invention, the inhibitors targeting KRAS G12D gene mutations include: MRTX1133, HRS-4642, AZD0022, and LY3962673.

[0024] In some embodiments of the present invention, the inhibitors against KRAS G12V gene mutations include: AFNT-211, NW-301V, and CRTKVA11. In some embodiments of the present invention, the KRAS inhibitor includes adagracib.

[0025] In some embodiments of the present invention, the tumor includes non-small cell lung cancer and pancreatic cancer.

[0026] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0027] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: propellants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0028] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0029] In some embodiments of the present invention, the dosage form of the product includes one of the following: powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0030] In some embodiments of the present invention, the product is administered via the gastrointestinal tract or non-gastrointestinal route.

[0031] In some embodiments of the present invention, the gastrointestinal administration includes one of oral administration, sublingual administration, and rectal administration.

[0032] In some embodiments of the present invention, the non-gastrointestinal administration includes one of intravenous injection, subcutaneous injection, and mucosal administration.

[0033] In some embodiments of the present invention, the product is applied to mammals, including but not limited to: humans, mice, rats, pigs, cattle, sheep, horses, monkeys, and rabbits.

[0034] In some embodiments of the invention, the mammal includes humans.

[0035] A second aspect of the invention provides the use of HS2ST1 inhibitors and KRAS inhibitors in the preparation of medicaments for treating tumors.

[0036] In some embodiments of the present invention, the KRAS inhibitor includes an inhibitor targeting KRAS gene mutations or a broad-spectrum KRAS inhibitor.

[0037] In some embodiments of the present invention, the tumor includes non-small cell lung cancer and pancreatic cancer.

[0038] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0039] A third aspect of the present invention provides a pharmaceutical composition comprising an HS2ST1 inhibitor and a KRAS inhibitor.

[0040] The HS2ST1 inhibitor includes at least one of the following: (a1) Substances that inhibit the activity of HS2ST1 protein; (a2) Substances that reduce the content of HS2ST1 protein; (a3) Substances that knock out the HS2ST1 gene; (a4) Substances that inhibit the expression of the HS2ST1 gene.

[0041] In some embodiments of the present invention, the HS2ST1 inhibitor includes nucleic acid molecules, protein molecules, and small molecule compounds.

[0042] In some embodiments of the present invention, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide.

[0043] In some embodiments of the present invention, the KRAS inhibitor includes an inhibitor targeting KRAS gene mutations or a broad-spectrum KRAS inhibitor.

[0044] In some embodiments of the present invention, the KRAS inhibitor includes adagracib.

[0045] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0046] The beneficial effects of this invention are: Currently, over 50% of patients with KRAS inhibitor resistance face the clinical challenge of unclear mechanisms and a lack of effective intervention targets. This invention identifies HS2ST1 as a key regulatory target for KRAS inhibitor resistance for the first time. Studies have found that HS2ST1 expression is significantly upregulated in KRAS inhibitor-resistant cells; functional experiments confirm that targeted intervention with HS2ST1 can effectively enhance the efficacy of KRAS inhibitors and reverse their resistance phenotype, providing a new strategy and potential therapeutic direction for overcoming KRAS-targeted therapy resistance. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results show the upregulation of HS2ST1 expression in lung adenocarcinoma, pancreatic cancer, and colorectal cancer with high KRAS mutations. Specifically: (A) HS2ST1 mRNA expression levels in tumor tissues and normal tissues of patients with lung adenocarcinoma, pancreatic cancer, and colorectal cancer in the TCGA database; (B) Western blot detection of HS2ST1 protein expression levels in cancerous and adjacent tissues of 8 patients with lung adenocarcinoma. Figure 2To evaluate the sensitivity of KRAS G12C mutant cells to adagogracib, the following was performed: A) MTT assay was used to detect the adagogracib IC50 of four KRAS G12C mutant cell lines: H2030, Calu1, Mia paca2, and H358. 50 IC50 values ​​(μM); BC) MTT assay of the IC50 values ​​of Calu1 and Mia paca2 parental cells (PR) and their acquired resistant strains (AR) against adagrasib. 50 Value (μM); D) Western Blot analysis of the protein expression differences of HS2ST1 in Calu1 and Mia paca2 PR and AR; E) RT-qPCR detection of the knockdown efficiency of HS2ST1 knockdown in drug-resistant strains H2030, Mia paca2 AR, and Calu1 AR (μM); p <0.05 is *; p <0.05 is ** p <0.001 is ***).

[0048] Figure 3 To investigate whether knocking down HS2ST1 enhances the drug sensitivity of KRAS G12C mutant cells to adagrasibuline, the following methods were employed: (a) Clone formation assay was used to verify the inhibitory effect of HS2ST1 knockdown on the proliferation of H2030, Mia paca2 AR, and Calu1 AR cells under both drug-treated and untreated conditions; (b) MTT assay was used to detect the IC50 of adagrasibuline in the three drug-resistant cell lines (H2030, Mia paca2 AR, and Calu1 AR) after HS2ST1 knockdown. 50 Trends in change.

[0049] Figure 4 To investigate the effect of HS2ST1 overexpression on reducing the drug sensitivity of KRAS G12C mutant cells to adagrasib, the following assays were performed: AC) colony formation assay to analyze the promoting effect of HS2ST1 overexpression on cell proliferation in Calu1, Mia paca2, and H358 cells under adagrasib treatment; DF-MTT assay to detect the induced adagrasib IC50 in these cells due to HS2ST1 overexpression. 50 An upward trend.

[0050] Figure 5The results show the effects of HS2ST1 on the expression levels of p-ERK and p-AKT, including: A) Western blot analysis of the effect of HS2ST1 knockdown on p-ERK and p-AKT expression levels in the H2030 cell line; B) Western blot analysis of the effect of HS2ST1 knockdown on p-ERK and p-AKT expression levels in Calu1 AR cells after treatment with 1 μM adagraciab; and C) Western blot analysis of the effect of HS2ST1 overexpression on p-ERK and p-AKT expression levels in Calu1 AR and Mia paca2 AR cells after treatment with adagraciab at different concentrations for 24 hours.

[0051] Figure 6 To reveal the regulatory role of HS2ST1 expression level on the efficacy of adagrasib using a nude mouse subcutaneous tumorigenesis model ( p <0.05 is *; p <0.05 is ** p <0.001 indicates ***) Results, where: AC = changes in tumor volume (B) and tumor weight (C) after 22 days of adagrasib treatment with subcutaneously injected H2030-knockdown HS2ST1 (sh-HS2ST1) cells (compared to the control group H2030 V), and visually displays tumor morphology (A); DF = changes in tumor volume (E) and tumor weight (F) after 22 days of adagrasib treatment in the model of injecting Calu1-overexpressing HS2ST1 (OE HS2ST1) cells (compared to the control group Calu1 V), and visually displays tumor morphology (D). Detailed Implementation

[0052] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0053] Example 1 This invention, through systematic analysis of RNA sequencing data from patients with lung adenocarcinoma, pancreatic cancer, and colorectal cancer in the TCGA database, found that HS2ST1 mRNA was significantly upregulated in these malignant tumor tissues with high-frequency KRAS mutations.

[0054] 1. Experimental Methods Western blotting was used to analyze the differences in HS2ST1 protein levels between tumor tissues and paired adjacent normal tissues of NSCLC patients, as well as the expression of proteins such as p-ERK and p-AKT in drug-resistant cells.

[0055] Sample preparation: ① Tissue samples: The experiment was based on specimens from 8 clinical NSCLC patients (including tumor tissue and paired adjacent normal tissue; all samples were surgically removed from the First Affiliated Hospital of Sun Yat-sen University). The tissues were ground into a suspension using a mortar and pestle, washed twice with PBS, incubated at 37°C for 5 minutes with added red blood cell lysis buffer, washed once with PBS, and 200 μL of Sample Buffer was added.

[0056] ② Cell sample: Take tumor cells with a growth density of 80%, wash them once with PBS, and add an appropriate amount of Sample Buffer.

[0057] Experimental procedure: (1) The above sample cells were disrupted by sonication. The protein concentration was determined using the BCA protein assay kit. Protein denaturing agent was added at a ratio of 20:1. The mixture was heated in a metal bath at 100 degrees Celsius for 5 minutes. 40 μg of protein sample was taken and the volume was made up to 40 μL with Sample Buffer for loading. (2) Add the sample from (1) to the SDS-PAGE gel in sequence, and perform SDS-PAGE electrophoresis at 100V for 120 minutes. After electrophoresis, transfer the PVDF membrane to a wet electrophoresis apparatus at 300mA for 150 minutes, and then remove the PVDF membrane. Block with 5% skim milk for 1 hour, then incubate with primary antibody overnight on a shaker at 4 degrees Celsius.

[0058] (3) Add horseradish peroxidase-labeled secondary antibody of the corresponding species and incubate at room temperature for 1 hour. Wash three times with TBST buffer, add ECL chemiluminescence kit in a dark environment, and cover the X-ray film in time. Determine the X-ray film covering time according to the luminescence intensity, and then develop the film.

[0059] The above reagent formulations or sources: Sample Buffer: 1g sodium dodecyl sulfate (SDS); 9ml ultrapure water; 1ml β-mercaptoethanol; Protein denaturing agent: 2ml 0.5M Tris-HCl (pH 6.8); 2.5ml glycerol; 4ml 10% SDS; 1ml 1% bromophenol blue; 1ml β-mercaptoethanol; Ultrapure water to a total volume of 10mL; TBST buffer: Dilute 100mL of 10×TBS buffer with ultrapure water to 1L, add 1ml Tween-20 and mix thoroughly to prepare 0.1% 1×TBST buffer, store at room temperature; BCA protein assay kit: purchased from Thermo Fisher Scientific, Rockford, IL; ECL chemiluminescence solution: purchased from Thermo Fisher Scientific, Rockford, IL; SDS-PAGE gel: purchased from Novizan, catalog number E303-01; PVDF membrane: purchased from Roche, Indianapolis, IN.

[0060] 2. Experimental Results like Figure 1 As shown in the AC, this result indicates that the HS2ST1 mRNA level in tumor tissues is higher than that in adjacent normal tissues, and consistent expression differences are observed in lung adenocarcinoma, pancreatic cancer, and colorectal cancer samples.

[0061] result Figure 1 As shown in Figure D, Western blotting experiments revealed that the expression level of HS2ST1 protein in tumor tissues of 6 / 8 NSCLC patients was significantly higher than that in paired adjacent normal tissues.

[0062] Example 2 This invention selected 293T cells and four tumor cell lines with primary KRAS G12C mutations, including H2030, Calu1, and H358 (lung adenocarcinoma) and Mia paca2 (pancreatic cancer). All five cell lines were identified and confirmed by the Short Tandem Repeat (STR) assay at the Forensic Identification Center of Sun Yat-sen University and verified to be consistent with standards, ensuring cell reliability.

[0063] 1. Experimental Methods IC detection by MTT (thiazolyl blue) method 50Logarithmic growth phase cells were seeded into 96-well plates (density: 3000-5000 / well). After 12 hours of adhesion, the medium was replaced with drug-containing medium. After 72 hours of drug treatment, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 6 hours. The medium was then removed, and 150 μL of DMSO was added to each well, followed by shaking to dissolve the precipitate. The OD value at 490 nm was measured using a microplate reader, and the cell viability (%) was calculated as follows: (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well) × 100%.

[0064] This invention uses shRNA (Short Hairpin RNA) for knockdown, specifically sh HS2ST1-1 and sh HS2ST1-2, which are double-stranded fragments formed by annealing sh HS2ST1-1-F and sh HS2ST1-1-R, and sh HS2ST1-2-F and sh HS2ST1-2-R, respectively. These double-stranded fragments are ligated into a pLVX restriction vector (pLVX restriction sites are BamHI and EcoRI), and after transformation, pLVX sh HS2ST1-1 and pLVX sh HS2ST1-2 are transfected into 293T cells using Lipo 3000. Viral fluid is collected after 48 hours and filtered through a 0.22 μM filter. The viral fluid is then used to infect H2030, Calu1 AR, and Mia paca2 AR cells. After selection with 2 μg / ml puro for 1-2 weeks, RNA is collected to assess the knockdown efficiency.

[0065] The shRNA sequence is as follows: shHS2ST1-1-F:5 -GATCCCCCATGTCATAAGGGATCCTATTGTTCAAGAGACAATAGGATCCCTTATGACATTTTTTG-3 (SEQ ID NO: 3); shHS2ST1-1-R: 5 -AATTCAAAAAATGTCATAAGGGATCCTATTGTCTCTTGAACAATAGGATCCCTTATGACATGGG-3 (SEQ ID NO: 4); shHSST1-2-F:5 -GATCCCCGGATTATGATGCCGCCCAAGTTTCAAGAGAACTTGGGCGGCATCATAATCCTTTTTG-3 (SEQ ID NO: 5); shHSST1-2-R: 5 -AATTCAAAAAGGATTATGATGCCGCCCAAGTTCTCTTGAAACTTGGGCGGCATCATAATCCGGG-3 (SEQ ID NO: 6).

[0066] in: shHS2ST1-1 sense seq: 5 -CATGTCATAAGGGATCCTATTG-3 (SEQ ID NO: 7); ShHS2ST1-2 sense seq: 5 -CGGATTATGATGCCGCCCAAGT-3 (SEQ ID NO: 8).

[0067] plasmid construction (1) sh HS2ST1-1-F, sh HS2ST1-1-R; sh HS2ST1-2-F, sh HS2ST1-2-R were sent to Guangzhou Ruibo Company to synthesize 20 μM primers, and the annealing reaction system (total 50 μL) was prepared according to the following system: F2μl; R2μl; 5×Annealing Buffer 10μl; ddH2O 36μl.

[0068] Repeat the mixing of the prepared annealing reaction buffer, briefly centrifuge, place above PCR, and run the following program: 90℃ for 4 min, 70℃ for 10 min, 90℃ for 4 min, 70℃ for 10 min, 37℃ for 15 min, 10℃ for 15 min.

[0069] (2) Enzyme digestion of vector The vector pLVX was double-digested with BamHI and EcoRI: the following system (50 μl) was used: BamHI: 2μl; EcoRI: 2μl; pLVX Vector: Xul(2μg); rCutsmart buffer 5μl; ddH2O up to 50 μl (all endonucleases and buffers were purchased from NEB). 37℃ for 2-4 hours.

[0070] After enzyme digestion, gel electrophoresis was performed, and the gel block was recovered using a gel recovery kit (Kangrun Company, catalog number D205).

[0071] (3) Connecting carrier, reaction system (10 μl) Vector: 1μl; Annealed product: 6 μl; 3×ligation high (purchased from NEB): 3μl; 16℃ for 2 hours.

[0072] (4) Transform E. coli competent cells DH5α with the ligation product. Transformed cells were cultured on ampicillin-resistant agar plates at 37°C. After about 14-16 hours, single bacterial colonies appeared on the plates. Single colonies were picked and sent to Guangzhou Ruibo Company for sequencing identification.

[0073] Construction of stable cell lines: (1) Lipofectamine 3000 (purchased from Thermo Fisher Scientific) was used to transfect 293T cells with a growth density of approximately 60%.

[0074] (2) 48 hours after transfection, the cell supernatant was taken out, filtered through a 0.22 μm filter, and added to the cells to be infected.

[0075] (3) Replace the culture medium with Puromycin at a concentration of 2 μg / ml 24 h after infection and continue culturing for 1-2 weeks (replace the culture medium with the drug every three days) until a large number of stable cells are screened out and the knockdown efficiency is verified.

[0076] RNA extraction and RT-qPCR (1) Once the cell density reaches approximately 80%, RNA is rapidly extracted using the Universal RNA Purification Kit (Catalog No.: EZB-RN4).

[0077] (2) Take 2 μg of RNA for reverse transcription using the GoScript™ kit (purchased from Promega). After reverse transcription, add 80 μL of enzyme-free water to dilute the cDNA.

[0078] (3) Prepare the following system (10 μl) F 1 μl; R 1 μl; 3 μl of cDNA; 2×SYBR Green mix (purchased from Novizan) 5 μl; 2 μl of enzyme-free water.

[0079] PCR reaction program: 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s, 60℃ for 60 s; cycle reaction conditions: 95℃ for 15 s, 60℃ for 60 s, for a total of 40 cycles, 95℃ for 15 s.

[0080] RT-qPCR result analysis formula: 2-[(Ct of genes)–(Ct of ACTB).

[0081] The qPCR primer sequences used in this invention are as follows: H-HS2ST1-qPCR-F:5 -GACACGAAGTCCGAGAAATTGA-3 (SEQ ID NO: 9); H-HS2ST1-qPCR-R:5 -ACAGGTCATAGGCGATATTGGTA-3 (SEQ ID NO: 10); H-ACTB-qPCR-F:5 -CATGTACGTTGCTATCCAGGC-3 (SEQ ID NO: 11); H-ACTB-qPCR-R:5 -CTCCTTAATGTCACGCACGAT-3 (SEQ ID NO: 12).

[0082] 2. Experimental Results Figure 2 The data in section A shows that Adagarasib has ICs for H2030, Calu1, H358, and Mia paca2. 50 The values ​​were approximately 4.189, 0.112, 0.008, and 0.192 μM, respectively. Therefore, H2030 was defined as a primary adagrasib-resistant strain, and Calu1, H358, and Mia paca2 were defined as adagrasib-sensitive strains. Next, the adagrasib-resistant cell lines Calu1 AR (Adagrasib resistance) and Mia paca2 AR were constructed using a concentration gradient method. Figure 2 As shown in Figure BC, the IC50 of Calu1AR is approximately 2.839 (μM), and the IC50 of Mia paca2AR is approximately 4.815 (μM). Their resistance index (RI = IC50 of resistant cell lines) is... 50 / IC of parental cell lines 50The RIs were approximately 25.35 and 25.07, respectively, with RIs both greater than 5, indicating that the acquired resistance strain of adagrasib was successfully constructed.

[0083] Figure 2 As shown in Figure D, Western blotting was used to detect the protein level of HS2ST1 in parental (PR) cells and drug-resistant strains. It was found that the expression level of HS2ST1 in AR cells was significantly increased compared with PR cells.

[0084] Figure 2 As shown in Figure E, the mRNA expression level of HS2ST1 in the sh HS2ST1 group was significantly lower than that in the V (blank control group) by RT-qPCR experiment, indicating that HS2ST1 was successfully knocked down in the three drug-resistant cell lines.

[0085] Example 3 1. Experimental Methods For the colony formation assay, logarithmically growing cells were seeded into 12-well plates (density: 1000 / well). After 24 hours of adhesion, the medium was replaced with drug-containing medium. Cells were cultured for 10-14 days (replacing with fresh drug-containing medium every 3 days) until visible colonies formed in the control group. Cells were fixed with methanol for 15 minutes and stained with 1% crystal violet overnight at room temperature. Residual dye was rinsed off with running water, and cells were air-dried at room temperature. Clonal morphology was recorded using a high-resolution scanner.

[0086] 2. Experimental Results Figure 3 As shown in the AC, under untreated conditions, knockdown of HS2ST1 had no significant effect on the proliferation of H2030, Calu1 AR, and Mia paca2 AR; conversely, when 1 μM adagrasib was administered, knockdown of HS2ST1 significantly reduced the number of colonies formed (compared to the drug-resistant control), indicating that knockdown of HS2ST1 enabled drug-resistant cells to regain drug sensitivity.

[0087] Figure 3 As shown in the middle DF, MTT assay revealed that knocking down HS2ST1 in three drug-resistant cell lines (H2030, Calu1 AR, and Mia paca2 AR) resulted in an IC50 concentration of 1,000 mg / dL. 50 The value was significantly reduced, partially reversing the drug resistance phenotype, further supporting the role of HS2ST1 as a drug resistance target.

[0088] Experiment Example 4 Figure 4 As shown in Figure AC, in adagaracilb-sensitive cell lines Calu1, Mia paca2, and H358, treatment with 0.25 μM, 0.4 μM, and 0.02 μM of the drug, respectively (concentration corresponding to the IC50 of each parent cell line), resulted in... 50 When the expression of HS2ST1 was twice the value, overexpression of HS2ST1 significantly increased the number of clones formed.

[0089] Figure 4 As shown in Figure DF, IC is detected by the MTT method. 50 Values ​​showed that overexpression of HS2ST1 in sensitive cells significantly improved IC50. 50 The above results indicate that HS2ST1 overexpression effectively induces drug resistance in sensitive strains and weakens the inhibitory effect of adagrasib.

[0090] Experimental Example 5 KRAS inhibitors (such as adagraxibu) exert their therapeutic effect by immobilizing KRAS in an inactivated state. However, resistant cells can reactivate downstream MAPK / ERK and PI3K / AKT signaling pathways through KRAS-dependent / independent pathways, leading to increased p-ERK and p-AKT levels and driving tumor cell proliferation, differentiation, and escape. This reactivation process is the core molecular mechanism of acquired resistance.

[0091] Figure 5 As shown in Figure A, Western blotting experiments revealed that knocking down HS2ST1 in H2030 cells, under untreated conditions, reduced the expression levels of p-ERK and p-AKT. This indicates that HS2ST1 is a key regulator for maintaining the activity of basal signaling pathways, and its inhibition can weaken the phosphorylation of downstream molecules. Figure 5 As shown in Figure B, the Calu1 AR resistant strain resisted the inhibitory effect of 1 μM adagrasibu on p-ERK and p-AKT (i.e., the drug could not significantly reduce their expression). However, after knocking down HS2ST1, the resistant strain lost its resistance—p-ERK and p-AKT expression was significantly reduced under the same drug treatment, indicating that HS2ST1 can mediate the reactivation of the signaling pathway. Figure 5 As shown in the CD, overexpression of HS2ST1 in the sensitive strains Calu1 and Mia paca2 significantly resisted the downregulation of p-ERK / p-AKT by high concentrations of adagraxibuvir (compared to empty vector control cells). This indicates that HS2ST1 drives drug resistance by maintaining p-ERK and p-AKT levels.

[0092] Experimental Example 6 Using a nude mouse subcutaneous tumorigenesis model, we evaluated the role of HS2ST1 as a key target for adagracilistylus resistance in vivo.

[0093] 1. Experimental Methods The female Balb / c-nu mice used in this experiment were purchased from Guangdong Yaokang Biotechnology Co., Ltd. All mice were housed under specific pathogen-free conditions and used according to protocols approved by the Animal Welfare Institution Care and Use Committee of Sun Yat-sen University. Mice were initially randomly grouped by age and weight, and were 4-6 weeks old at the time of injection.

[0094] H2030 group: divided into H2030 V (empty control group) and H2030 sh-HS2ST1 (HS2ST1 knockdown group), 5 mice in each group, each mouse was subcutaneously injected with 3×10 6 Tumor cells; Calu1 group: divided into Calu1 V (empty vector control group) and Calu1 OEHS2ST1 (HS2ST1 overexpression group), 4 mice in each group, each mouse was injected with 3×10 6 Tumor cells were identified. After tumor formation, adagrasib was administered: 30 mg / kg in the H2030 group and 20 mg / kg in the Calu1 group. The medication was administered by gavage every 3 days for a total of 21 days, with tumor volume monitored every 3 days.

[0095] 2. Experimental Results Figure 6 As shown in the AC study, adagrasib (30 mg / kg) developed resistance quickly in mice subcutaneously injected with H2030 empty vector cells, and tumor growth was not effectively inhibited. In contrast, knocking down HS2ST1 significantly enhanced the efficacy of adagrasib: both tumor volume and weight were significantly reduced. This result confirms that targeting and inhibiting HS2ST1 can effectively reverse the resistance phenotype of adagrasib.

[0096] Figure 6 As shown in the DF, adagraxib (20 mg / kg) effectively inhibited tumor growth in mice subcutaneously injected with Calu1 empty vector cells, with no significant increase observed within 21 days. However, overexpression of HS2ST1 significantly reduced the drug's efficacy: both tumor volume and weight increased significantly. This indicates that HS2ST1 is a key target for inducing the transformation of tumor cells from a sensitive to a drug-resistant state under the action of adagraxib.

[0097] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of a HS2ST1 inhibitor in the preparation of a medicament for treating a KRAS inhibitor-resistant tumor.

2. The use according to claim 1, wherein: the HS2ST1 inhibitor comprises at least one of: (a1) a substance that inhibits the activity of a HS2ST1 protein; (a2) a substance that reduces the content of a HS2ST1 protein; (a3) a substance that knocks out a HS2ST1 gene; (a4) a substance that inhibits the expression of a HS2ST1 gene.

3. The use according to claim 2, wherein: the HS2ST1 inhibitor comprises a nucleic acid molecule, a protein molecule, or a small molecule compound; preferably, the nucleic acid molecule is a microRNA, an siRNA, an shRNA, a dsRNA, an sgRNA, and / or an antisense oligonucleotide; preferably, the protein molecule is a specific antibody of HS2ST1; preferably, the small molecule compound is a small molecule compound that inhibits HS2ST1.

4. The use according to claim 3, wherein: the sequence of the shRNA is as shown in SEQ ID NO: 7 or 8.

5. The use according to claim 1, wherein: the KRAS inhibitor comprises an inhibitor against a KRAS gene mutation or a broad-spectrum KRAS inhibitor; the KRAS gene mutation comprises KRAS G12C, KRAS G12D, or KRAS G12V.

6. The use according to claim 2, wherein: the KRAS inhibitor comprises adagrasyl.

7. The use according to claim 1, wherein: the medicament comprises a pharmaceutically acceptable excipient.

8. The use according to claim 1, wherein: the dosage form of the medicament comprises a gastrointestinal administration dosage form or a non-gastrointestinal administration dosage form.

9. Use of a combination of a HS2ST1 inhibitor and a KRAS inhibitor in the preparation of a medicament for treating a tumor.

10. A pharmaceutical composition, comprising: the pharmaceutical composition comprises a HS2ST1 inhibitor and a KRAS inhibitor; preferably, the KRAS inhibitor comprises adagrasyl.

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