Use of gpx4 inhibitor in combination with a parp inhibitor in the preparation of an anti-tumor drug

Abstract

The application discloses application of a GPX4 inhibitor combined with a PARP inhibitor in preparation of an antitumor drug, and is particularly directed to tumors with BRCA1 mutation or deletion. The GPX4 inhibitor reverses the tolerance of tumors with BRCA1 mutation or deletion to ferroptosis, and produces a synergistic effect with the PARP inhibitor. The application effectively solves the problems of drug resistance of the PARP inhibitor and tolerance of tumors with BRCA1 mutation or deletion to ferroptosis, and the application shows the advantages of high efficiency and low toxicity in vitro and in vivo, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the application of a GPX4 inhibitor combined with a PARP inhibitor in the preparation of antitumor drugs, and more particularly to the application of a GPX4 inhibitor combined with a PARP inhibitor in the preparation of anti-BRCA1 mutant or deleted tumor drugs. Background Technology

[0002] Poly(ADP-ribose) polymerase (PARP) is an enzyme that catalyzes the transfer of ADP-ribosyl groups to receptor proteins. It is primarily located in the nucleus of eukaryotic cells and participates in the poly-ADP-ribosylation modification of nuclear proteins such as histones, topoisomerases, and DNA polymerases. The PARP family comprises 18 subtypes, with PARP1 being the most abundant and studied. PARP1 recognizes and binds to DNA break sites via its N-terminus, playing a crucial role in base excision repair and single-strand break repair. As a DNA single-strand break repair protein, PARP inhibition leads to the accumulation of unrepaired single-strand breaks, which subsequently transform into lethal DNA double-strand breaks. DNA double-strand breaks formed after PARP inhibitor treatment are highly dependent on homologous recombination for repair; therefore, PARP inhibitors interact with tumor cells exhibiting homologous recombination repair deficiency (HRD) to form synthetic lethality. Mutations in the tumor suppressor genes BRCA1 and BRCA2, which are homologous recombination repair molecules, lead to HRD deficiency. Therefore, PARP inhibitors are widely used clinically to treat BRCA1 / 2-mutated tumors, including ovarian cancer, breast cancer, prostate cancer, and pancreatic cancer. Currently, four PARP inhibitors have been approved for clinical use, including olaparib, rucaparib, niraparib, and talazoparib, which have become first-line maintenance therapy drugs for BRCA1 / 2-mutated ovarian cancer.

[0003] BRCA1 and BRCA2 genes are important tumor suppressor genes in the human body and are crucial biomarkers for assessing the risk of breast cancer, ovarian cancer, and other related tumors. The human BRCA1 gene is located on chromosome 17 q21, approximately 81 kb in size, encoding a BRCA1 protein of approximately 220 kDa, consisting of 1863 amino acid residues. The human BRCA2 gene, located on the long arm of chromosome 13, region 12, consists of 27 exons and encodes a BRCA2 protein of 3418 amino acids. Both BRCA1 and BRCA2 proteins play important regulatory roles in multiple steps of heart rate repair (HR) and are considered key regulatory molecules for HR repair. Deletion or mutation of BRCA1 or BRCA2 leads to severely impaired HR repair. BRCA1 / 2 gene mutations are very common in human tumors; approximately 20-30% of ovarian or breast cancer tissues show BRCA1 or BRCA2 mutations. As targeted therapies for BRCA1 / 2-mutant tumors, PARP inhibitors are significantly more effective in patients with BRCA2-mutant tumors than in those with BRCA1-mutant tumors. Some patients with BRCA1-mutated tumors are not very sensitive to PARP inhibitors, and the problem of PARP inhibitor tolerance in BRCA1-mutated patients has become widespread in clinical practice. Therefore, there is an urgent clinical need for a new treatment strategy to reverse PARP inhibitor tolerance. Summary of the Invention

[0004] Purpose of the invention: The present invention aims to provide the application of a GPX4 inhibitor combined with a PARP inhibitor with synergistic effects in the preparation of antitumor drugs.

[0005] Technical solution: The GPX4 inhibitor combined with the PARP inhibitor described in this invention is used in the preparation of anti-tumor drugs.

[0006] Preferably, the drug is an anti-BRCA1 mutated or deleted tumor drug.

[0007] Further preferably, the drug is an anti-BRCA1-mutated or deleted ovarian cancer, breast cancer, prostate cancer, or pancreatic cancer, and more preferably BRCA1-mutated or deleted ovarian cancer.

[0008] This invention utilizes tumor cell models, ovarian cancer organoids, and ovarian cancer animal models to discover that the combined use of GPX4 inhibitors and PARP inhibitors can produce synergistic anti-tumor effects and synergistically promote ferroptosis in ovarian cancer cells.

[0009] Preferably, the GPX4 inhibitor is selected from hexamethylmelamine (Altretamine), ML210, ML162, RSL3, and FIN56, and more preferably from hexamethylmelamine (Altretamine) and ML210.

[0010]

[0011] Preferably, the PARP inhibitor is selected from olaparib, rucaparib, niraparib, talazoparib, pamiparib, fluzoparib, and veliparib, and more preferably olaparib, rucaparib, niraparib, and talazoparib.

[0012] The preferred combination is a GPX4 inhibitor (ML210) with a PARP inhibitor (olaparib), or a GPX4 inhibitor (Altretamine) with a PARP inhibitor (olaparib, rucaparib, niraparib, taraparib).

[0013] The GPX4 inhibitor described in this invention is used as a sensitizer in the preparation of PARP inhibitors against tumors with BRCA1 mutations or deletions.

[0014] Preferably, the GPX4 inhibitor reverses the tolerance of ferroptosis in BRCA1-mutated or deleted tumors.

[0015] Further preferably, the BRCA1 promotes tumor sensitivity to ferroptosis by polyubiquitination modification of GPX4.

[0016] Ferroprelation is an iron-dependent form of cell death caused by lethal lipid peroxidation, characterized by cell contraction and increased mitochondrial membrane density. Ferroprelation is controlled by intracellular oxidative and antioxidant systems, among which the selenium-containing antioxidant enzyme glutathione peroxidase 4 (GPX4) is considered a central inhibitory factor of ferroprelation. Ferroprelation plays an important role in various physiological and pathological processes, including neurodegenerative diseases, acute renal failure, cardiac injury, and cancer. As a regulatory form of cell death, susceptibility to ferroprelation is highly correlated with tumor progression and treatment resistance.

[0017] Currently, the correlation between ferroptosis and tumors with BRCA1 mutations or deletions has not been studied. This invention utilizes proteomics, molecular biology, and cell biology experiments to discover that BRCA1 interacts with GPX4 and directly catalyzes the K6-linked polyubiquitination modification of GPX4, promoting GPX4 degradation and thereby enhancing ferroptosis sensitivity. This invention confirms that tumors with BRCA1 mutations or deletions develop tolerance to ferroptosis by upregulating GPX4 protein levels.

[0018] There is no existing research on the correlation between the BRCA1 tumor suppressor gene and the regulation of GPX4 protein levels. This invention is the first to demonstrate the use of a combination of GPX4 inhibitors and PARP inhibitors in the treatment of BRCA1-mutant ovarian cancer. It can exert a synergistic anti-tumor effect by increasing the ferroptosis sensitivity of ovarian cells, providing a new approach and means for the effective treatment of ovarian cancer and showing good application prospects.

[0019] The pharmaceutical compositions of the present invention, which use GPX4 inhibitors and PARP inhibitors as active ingredients, or pharmaceutical compositions using GPX4 inhibitors and PARP inhibitors as active ingredients respectively, are used in combination in the preparation of antitumor drugs.

[0020] The active ingredient contained in the pharmaceutical composition may also be a pharmaceutically acceptable salt of the GPX4 inhibitor or PARP inhibitor.

[0021] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0022] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0023] The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

[0024] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0025] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0026] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0027] Preferably, the dosage ratio of the GPX4 inhibitor to the PARP inhibitor is (0.25-50):(0.01-50), specifically (0.5-6):(0.01-10), (1-50):(0.01-10), (1-50):(5-25), (0.25-30):(0.4-50); more preferably, the dosage ratio is 1:1 or 3:10.

[0028] The preferred combination therapy is the use of a GPX4 inhibitor (ML210) in combination with a PARP inhibitor (olaparib) at a dosage ratio of (0.5–6):(0.01–10) or (0.25–30):(0.4–50); or the use of a GPX4 inhibitor (Altretamine) in combination with a PARP inhibitor (olaparib, rucaparib, niraparib, taprazolepanib) at a dosage ratio of (1–50):(0.01–10) or (1–50):(5–25). Specifically, the dosage ratio of the combination of the GPX4 inhibitor (Altretamine) with the PARP inhibitor (olaparib, rucaparib, taprazolepanib) is (1–50):(0.01–10); and the dosage ratio of the combination of the GPX4 inhibitor (Altretamine) with the PARP inhibitor (niraparib) is (1–50):(5–25). More preferred combination therapy is the combination of GPX4 inhibitor (ML210) and PARP inhibitor (olaparib) at a dosage ratio of 3:10; or the combination of GPX4 inhibitor (Altretamine) and PARP inhibitor (olaparib) at a dosage ratio of 1:1.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] It effectively solves the problem of resistance to PARP inhibitors and effectively reverses the tolerance of ferroptosis in BRCA1-mutated or deleted tumors, exerting a synergistic anti-tumor effect. At the same time, it shows the advantages of high efficiency and low toxicity in both in vivo and in vitro, and has good application prospects. Attached Figure Description

[0031] Figure 1 Results showing that BRCA1 downregulates GPX4 protein levels via polyubiquitination modification;

[0032] Figure 2 Knocking down GPX4 can reverse the ferroptosis tolerance induced by BRCA1 deficiency in PARP inhibitors.

[0033] Figure 3The study investigated the effects of combined use of GPX4 inhibitors (ML210), PARP inhibitors (olaparib), GPX4 inhibitors (ML210) + PARP inhibitors (olaparib), GPX4 inhibitors (Altretamine), PARP inhibitors (olaparib, rucaparib, niraparib, taraparib), and GPX4 inhibitors (Altretamine) + PARP inhibitors (olaparib, rucaparib, niraparib, taraparib) on cell proliferation.

[0034] Figure 4 The study investigated the effects of GPX4 inhibitor (ML210), PARP inhibitor (olaparib), and the combination of GPX4 inhibitor (ML210) and PARP inhibitor (olaparib) on the viability of organoids from patient-derived ovarian cancer.

[0035] Figure 5 To analyze the effects of GPX4 inhibitor (ML210), PARP inhibitor (olaparib), GPX4 inhibitor (ML210) + PARP inhibitor (olaparib), and GPX4 inhibitor (Altretamine), PARP inhibitor (olaparib), and GPX4 inhibitor (Altretamine) + PARP inhibitor (olaparib) on tumor growth in nude mice in tumorigenesis experiments;

[0036] Figure 6 The results of the analysis of the toxic side effects of GPX4 inhibitor (ML210), PARP inhibitor (olaparib), and the combination of GPX4 inhibitor (ML210) and PARP inhibitor (olaparib) in mice. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the embodiments.

[0038] Example 1: Study on the downregulation of GPX4 protein levels by BRCA1 through polyubiquitination modification

[0039] I. Experimental Design

[0040] This embodiment first verifies the interaction between BRCA1 and GPX4 through immunoprecipitation (IP) and adjacent linkage (PLA) experiments, then verifies BRCA1 ubiquitination modification of GPX4 through exogenous and endogenous IP, and finally verifies the protein expression level of GPX4 by knocking out and knocking down BRCA1.

[0041] II. Experimental Procedure

[0042] The experiments and methods involved in this embodiment are as follows:

[0043] 1. Immunoprecipitation (IP) assay

[0044] (1) Collect cells;

[0045] (2) Add EBC buffer (50mM Tris-HCl pH=7.6-8.0, 120mM NaCl, 0.5% NP-40, 1mM EDTA, 50mM NaF, 1mM Na3VO4 and 1mM β-mercaptoethanol) and lyse the cells by sonication;

[0046] (3) Centrifuge at 4℃, 12000r for 20min;

[0047] (4) Transfer the supernatant to a new EP tube, mix well, take 40 μL as input, add loading buffer, boil at 100℃ for 6 min; the remaining sample with the target antibody and protein A / G agarose beads, rotate and incubate overnight at 4℃; if it is a Flag immunoprecipitation, the remaining sample with anti-flag magnetic beads, rotate and incubate overnight at 4℃.

[0048] (5) On the second day, centrifuge at 1000g for 1 min at 4℃, discard the supernatant, add pre-cooled EBC buffer to wash the beads 3 times, and discard the supernatant.

[0049] (6) Add an appropriate amount of 2x loading buffer and heat at 100℃ for 10 minutes;

[0050] (7) Western blot analysis of proteins in the immunoprecipitation complex.

[0051] 2. Adjacency Linkage (PLA)

[0052] according to PLAFluorescence Protocol In Situ Red Mouse / Rabbit Starter Kit (DUO92101) - Follow the instructions in the kit manual:

[0053] (1) Spread the cells onto the glass slide one day in advance;

[0054] (2) Wash with PBS for 2 min, fix cells with 4% paraformaldehyde at room temperature for 15 min and then aspirate;

[0055] (3) Add 100 μl of 0.2% Triton X-100 to each well and treat the cells at room temperature for 10 min;

[0056] (4) Discard 0.2% Triton X-100, wash twice with PBS for 2 min each time;

[0057] (5) Add 40 μl of Duolink PLABlocking Buffer to block, and incubate at 37°C for 1 h;

[0058] (6) Discard the Blocking Buffer, add the primary antibody, and incubate at 37°C for 2 hours or 4°C overnight;

[0059] (7) Remove the primary antibody, add 1xWash Buffer A and wash twice at room temperature for 5 min each time;

[0060] (8) Discard washbufferA, add PLAprobe solution (40μL = 8μL of PLAprobe MINUSstock + 8μL of PLAprobe PLUSstock + 24μL of Antibody Diluent), and incubate at 37℃ for 1h;

[0061] (9) Discard the PLAprobe solution, add 1x Wash Buffer A and wash twice at room temperature for 5 minutes each time;

[0062] (10) Discard washbufferA, add 1x ligation buffer (1μL of ligase + 39μL of the 1x ligation buffer), and incubate at 37℃ for 30 min;

[0063] (11) Discard the ligation buffer, add 1xWash BufferA and wash twice at room temperature for 5 minutes each time;

[0064] (12) (Avoid light) Discard washbuffer A, add Amplification buffer, and incubate at 37°C for 100 min;

[0065] (13) Discard the buffer, add 1x Wash Buffer B and wash twice at room temperature for 10 minutes each time;

[0066] (14) Discard the buffer and add 0.01xWash Buffer B to wash for 1 minute;

[0067] (15) Add DAPI to each well for mounting and stain in the dark for 15 min;

[0068] (16) Take photos and analyze them.

[0069] 3. Ubiquitination analysis

[0070] In vivo experimental protocol: Flag-tagged GPX4 and HA-tagged Ub were co-transfected into cells. Before cell collection, cells were treated with MG132 (5 μM) for 6 h. 48 h post-transfection, cells were collected to prepare a protein suspension, which was then incubated overnight at 4°C using ANTI-FLAG M2 Beads. After thorough washing with EBC buffer, cells were resuspended in 2×SDS sample buffer and boiled at 100°C for 5 min. Finally, GPX4 ubiquitination was detected by Western blot using HA antibody.

[0071] In vitro experimental protocol: Heterodimers of WT or I26A and BARD1 were purified from transfected 293T cells using anti-Flag magnetic beads and 3X flag elution peptide. His-GPX4 (1 μg) was incubated in a reaction mixture containing 0.1 μg E1, 0.5 μg UBCH5C, 2 μg Ub, flag-BRCA11-303, and BARD1 (50 mM Tris-HCl pH 7.5, 120 mM NaCl, 0.5 mM MTT, 5 mM ATP, 5 mM MgCl2, 2 μg NaF, and 5 μM ZnCl2). After incubation at 37°C for 3 h, SDS-PAGE sample buffer was added, and the mixture was boiled at 100°C for 6 min. GPX4 ubiquitination was detected by Western blot using anti-Ub antibody.

[0072] 4. Western blot experiment

[0073] (1) Prepare the appropriate concentration of SDS-PAGE separating gel and stacking gel according to the requirements;

[0074] (2) Sample loading: Add protein markers and samples to wells according to the experimental design;

[0075] (3) First, run the sample to the separating gel with 80V, then switch to 120V to continue electrophoresis. According to the protein marker, once the target protein has run away, you can stop electrophoresis and transfer the membrane.

[0076] (4) Prepare a tray with sufficient transfer solution, place the transfer clamp with the transparent side of the transfer clamp facing down, and place a sponge pad, filter paper, and a PVDF membrane of appropriate size (pre-activated by soaking in methanol for more than 10 seconds) from bottom to top, and use a small roller to push away air bubbles.

[0077] (5) Take out the separating gel, place it flat on the PVDF membrane, and place filter paper and sponge pad on it in sequence. After using the roller to remove the air bubbles, close the transfer clamp and place it in the transfer tank. Pay attention to the placement direction, that is, the transparent side of the transfer clamp faces the red side of the transfer tank.

[0078] (6) Place the transfer tank into the electrophoresis apparatus in the corresponding orientation, pour in sufficient transfer buffer and place an ice pack for cooling inside, then cover the electrophoresis tank. Immerse the entire electrophoresis tank in a mixture of ice and water for cooling;

[0079] (7) Start by transferring the membrane at a constant voltage of 100V for 120 minutes. The specific transfer time can be adjusted according to the molecular weight of the protein. The larger the molecular weight, the longer the transfer time.

[0080] (8) After the transfer time is up, take out the PVDF membrane from the transfer tank and put it into the prepared 5% skim milk, and place it on a shaker and shake it slowly for 1 hour.

[0081] (9) After blocking, wash the milk with PBST, put the target band into the corresponding primary antibody prepared with 5% BSA solution (generally diluted at a ratio of 1:1000), and incubate overnight at 4°C on a shaker.

[0082] (10) After incubation, remove the strips and wash them with PBST 4 times, 5 min each time;

[0083] (11) Select the corresponding species of secondary antibody and dilute it with 5% skim milk to a suitable concentration. Place the cleaned PVDF membrane into the prepared secondary antibody and incubate it on a shaker at room temperature for 1 hour.

[0084] (12) After incubation, wash with PBST four times for five minutes each time. After wiping off the attached liquid, add ECL developer solution, which is a 1:1 mixture of solution A and solution B, to the strip. Place the strip in the dark chamber of the developing instrument for exposure and use a fully automated chemiluminescence image analysis system for photographic analysis.

[0085] III. Experimental Results

[0086] First, the interaction between BRCA1 and GPX4 was verified in A2780 cells via intracellular injection (IP). The results showed that BRCA1 and GPX4 interact in A2780 cells. Figure 1 A). Similarly, the PLA signal of BRCA1 / GPX4 interaction could be clearly detected by the proximity connection assay (PLA), and their interaction could be observed in both the cytoplasm and nucleus of A2780 cells, further verifying the correlation between BRCA1 and GPX4. Figure 1 B).

[0087] Then, GPX4 polyubiquitination was verified by co-transfecting flag-GPX4 and HA-Ub into UWB1.289 and UWB-BRCA1 cells. The results showed that the GPX4 ubiquitination level detected in UWB-BRCA1 cells was higher than that in UWB.1289 cells. Figure 1C). Similarly, using purified WT, E2-binding defective mutants (I26A) flag-BRCA1-303 and flag-BARD1 proteins, in vitro ubiquitination experiments revealed that the I26ABRCA1-303 / BARD1 complex could catalyze GPX4 polyubiquitination in the presence of E1 / E2 / Ub. Figure 1 D), which indicates that BRCA1 is an E3 ligase of GPX4.

[0088] Finally, Western blot experiments showed that GPX4 protein expression was significantly increased in A2780 cells with BRCA1 knockout and in SKOV3 and OVCAR-3 cells with BRCA1 knockdown. Figure 1 E, Figure 1 F).

[0089] Example 2: Study on how knocking down GPX4 can reverse BRCA1 deficiency-induced ferroptosis tolerance

[0090] I. Experimental Design

[0091] This embodiment analyzed the sensitivity of UWB1.289 cells to ferroptosis by adding different concentrations of the PARP inhibitor olaparib, using flow cytometry. Then, it analyzed the level of ferroptosis in cancer tissues before and after niraparib treatment in ovarian cancer samples that had relapsed following PARP inhibitor treatment. Finally, it analyzed the effect of GPX4 knockdown on PARP inhibitor-induced ferroptosis in BRCA1-deficient cells using clonogenic assays and flow cytometry.

[0092] II. Experimental Procedure

[0093] The experiments and methods involved in this example are as follows:

[0094] 1. Flow cytometry detection of lipid peroxidation

[0095] (1) Cells in the logarithmic growth phase were treated with different concentrations of reagents for 24 hours;

[0096] (2) Add 10 μM BODIPY 581 / 591C11 and incubate at 37℃ for 1 h;

[0097] (3) Digest cells with trypsin, centrifuge, and wash with PBS;

[0098] (4) Resuspend the cells in PBS and analyze the results by flow cytometry.

[0099] 2. Immunohistochemical assay (IHC)

[0100] (1) To dissolve the wax block on the slices, place the slices in a 60℃ oven for more than 30 minutes;

[0101] (2) After baking, the slices are placed in xylene to dewax four times, each time for 5 minutes;

[0102] (3) After removing the slices, place them in 100%, 90%, 70%, and 30% ethanol in sequence for 5 minutes each time to wash away the xylene.

[0103] (4) After removing the slices, wash them once with ddH2O for 5 minutes;

[0104] (5) Pour out ddH2O and replace it with a pre-prepared methanol-hydrogen peroxide mixture (ratio 9:1), shake on a shaker for 15 minutes;

[0105] (6) Pour out the mixture and wash once with ddH2O for 5 minutes;

[0106] (7) After placing the slices in the slice rack, put them into the prepared 1x sodium citrate antigen retrieval solution, bring to a boil on high heat, and then switch to low heat and cook for 10 minutes.

[0107] (8) After the sections have cooled to room temperature, wash them three times with PBS for 5 minutes each time;

[0108] (9) After removing the attached PBS from the slides, place them in a humidified chamber and add an appropriate amount of the prepared blocking solution (prepared according to the kit instructions using horse serum and Avidin reagent). Let stand at room temperature for 20 minutes.

[0109] (10) Remove the blocking solution from the slide, incubate with the corresponding antibody, and keep in a humidified chamber at 4°C overnight.

[0110] (11) Remove the incubated slices and wash them with PBS three times for 5 minutes each time;

[0111] (12) Add an appropriate amount of secondary antibody to the washed slices and incubate at room temperature in a humidified box for 20 min;

[0112] (13) Discard the secondary antibody, wash with PBS 3 times, 5 minutes each time;

[0113] (14) Remove as much PBS as possible from the slices, add Peroxidase, and incubate at room temperature in a humidified chamber for 15 min.

[0114] (15) Take out the slices and wash them with PBS 3 times for 5 minutes each time, and prepare the DAB solution according to the instructions;

[0115] (16) Remove the PBS attached to the slide, add DAB for visualization, and wash off the DAB quickly when the desired effect is achieved under the microscope to prevent over-staining;

[0116] (17) Stain the stained sections with hematoxylin in a humidified chamber for 50 seconds. The staining time can be adjusted according to the staining effect. After staining, wash with ddH2O for 5 minutes.

[0117] (18) After drying the slices in an oven at 60°C, seal them with neutral resin and store them in a cool, ventilated place. Take photos and analyze them.

[0118] 3. Cloning experiment

[0119] (1) 10 3 One cell was seeded into a 6-well plate;

[0120] (2) Grown for 7-14 days with or without olaparib until colonies appear;

[0121] (3) Discard the cell culture medium and wash three times with PBS;

[0122] (4) Fix the cells with formaldehyde for 15 min;

[0123] (5) Discard the formaldehyde and wash three times with PBS;

[0124] (6) Stain with crystal violet for 30 min;

[0125] (7) Discard the crystal violet and wash three times with PBS;

[0126] (8) Take photos and count the settlements.

[0127] III. Experimental Results

[0128] Flow cytometry analysis showed that in UWB1.289 cells, the level of lipid peroxidation increased with increasing olaparib concentration. Figure 2 A). IHC experiments also showed that after treatment with the PARP inhibitor (niraparib), the expression of the lipid peroxidation marker 4-HNE in the patient's cancer tissue was significantly increased, indicating that PARP inhibition can trigger ferroptosis in ovarian cancer cells. Figure 2 B). Then, GPX4 was knocked down in BRCA1-deficient cells. Colony formation assays and flow cytometry analysis revealed that GPX4 knockdown reversed BRCA1 deficiency-induced ferroptosis tolerance. Figure 2 C Figure 2 D).

[0129] Example 3: Bliss synergistic assay and clonogenic assay to study the synergistic antitumor effects of GPX4 inhibitors and PARP inhibitors in vitro.

[0130] I. Experimental Design

[0131] The antitumor effects were analyzed by administering GPX4 inhibitors (ML-210 or Altretamine), PARP inhibitors (olaparib, rucaparib, niraparib, taraparib) or a combination of both to cells, and by Bliss co-administration or clonogenic assay.

[0132] II. Experimental Procedure

[0133] 1. Bliss Collaborative Analysis

[0134] After digesting the cells with trypsin, 1000 cells / 100 μl of cell suspension was added to each well of a 96-well plate and allowed to adhere overnight. GPX4 inhibitor, PARP inhibitor, or a combination of both were added to each group and the cells were cultured for another 24 hours. CCK-8 was then added and the absorbance was measured using a microplate reader. Finally, Bliss co-processing was performed using GraphPad Prism.

[0135] 2. Cloning: 10 3 Cells were seeded into 6-well plates and grown for 7–14 days with 0.5 μM ML210, 0.5 μM olaparib, or a combination of both, until colonies appeared. Cells were then fixed with formaldehyde, stained with crystal violet, photographed, and counted.

[0136] III. Experimental Results

[0137] Bliss synergistic analysis showed that escalating doses of ML210 and escalating doses of olaparib synergistically inhibited cell growth. Figure 3 A); Simultaneously, escalating doses of another GPX4 inhibitor (Altretamine) synergistically inhibit cell growth with different types of PARP inhibitors (olaparib, rucaparib, niraparib, taprazole) Figure 3 B). Colony formation assays also showed that ML210 and olaparib synergistically inhibited cell growth significantly more effectively than either ML210 or olaparib alone. Figure 3 C).

[0138] Example 4: Study on the synergistic antitumor effect of patient-derived ovarian cancer organoids on GPX4 inhibitors and PARP inhibitors.

[0139] I. Experimental Design

[0140] In ovarian cancer organoids derived from patients, ML-210, olaparib, or a combination of both were administered, and the synergistic antitumor effect was calculated using the ZIP synergistic score.

[0141] II. Experimental Procedure

[0142] The patient-derived ovarian cancer organoid (PDO) model (BRCA1c.3756_3759del, PDO-ID:KOOA-009) was established by Beijing K2 Oncology Co., Ltd. (Beijing, China). Ovarian cancer tissues were collected from Yixing People's Hospital and approved by the Medical Ethics Committee of Yixing People's Hospital. Informed consent was obtained from patients for this study. Organoids were cultured in ovarian cancer organoid culture medium (#K2O-M-OA, K2 Oncology). After being seeded into 96-well plates, organoids were treated with different concentrations of ML210, olaparib, or a combination of both for 5 days. Organoid activity was detected by Cell titer-glo method (#G9683, Promega, Madison, WI), and synergistic effects were calculated by ZIP synergistic scoring.

[0143] III. Experimental Results

[0144] Patient-derived ovarian cancer organoids showed that ML210 and olaparib synergistically inhibited the activity of patient-derived ovarian cancer organoids, exerting a synergistic anti-tumor effect. Figure 4 A, Figure 4 B).

[0145] Example 5: Study on the synergistic anti-tumor effect of GPX4 inhibitors and PARP inhibitors in vivo in nude mouse tumorigenesis experiment.

[0146] I. Experimental Design

[0147] Ovarian cancer cells were subcutaneously inoculated into nude mice. When the tumors grew to a certain size, the mice were randomly divided into four groups: a control group, a GPX4 inhibitor (ML210) group, a PARP inhibitor (olaparib) group, and a GPX4 inhibitor (ML210) + PARP inhibitor (olaparib) group. Treatment efficacy was evaluated based on tumor size and body weight. Similarly, ovarian cancer cells were subcutaneously inoculated into nude mice. When the tumors grew to a certain size, the mice were randomly divided into four groups: a control group, a GPX4 inhibitor (Altretamine) group, a PARP inhibitor (olaparib) group, and a GPX4 inhibitor (Altretamine) + PARP inhibitor (olaparib) group. Treatment efficacy was evaluated based on tumor size and body weight.

[0148] II. Experimental Procedure

[0149] Six-week-old female BALB / c nude mice were housed in an SPF facility. 1×10 7 Cells were suspended in 100 μl PBS and subcutaneously injected into the right side of mice. When the tumor volume reached 50–100 mm, the cells were inoculated into the right side of mice. 3Mice were randomly divided into four groups: PBS (control) group, ML210 (30 mg / kg) group, olaparib (100 mg / kg) group, or a combination of both treatments. Another experimental group was randomly divided into PBS (control) group, Altretamine (100 mg / kg) group, olaparib (100 mg / kg) group, or a combination of both treatments. Body weight and tumor volume were measured every 3 days. Tumor volume was expressed as width (W) and length (L), i.e.: V = (W / L) * (L / L) * ... 2 The formula (×L) / 2 was used for calculation. Once the tumor reached a certain size, the mice were euthanized, the transplanted tumor was removed, and its size and weight were measured. After being photographed and labeled, the tumor was fixed in formalin for subsequent experiments.

[0150] III. Experimental Results

[0151] Compared to the control group, the tumor volume was slightly smaller in both the ML210 and olaparib groups. However, the ML210 + olaparib group showed a significantly higher tumor growth inhibition efficiency after combination therapy than either ML210 or olaparib alone, and the tumor weight in the combination therapy group was also significantly smaller than that in the single-drug therapy group. Figure 5 A). Meanwhile, the combination of another GPX4 inhibitor, Altretamine, and olaparib also showed significantly greater inhibitory efficacy against tumor growth compared to single-drug therapy. Figure 5 B) Throughout the treatment process, there were no significant differences in body weight changes among the control group, ML210 group, olaparib group, and ML210+olaparib group. Figure 6 A). HE experiments also showed that neither the single-drug group nor the combination drug group caused damage to the kidneys, spleen, lungs, and liver, indicating that ML210, olaparib, and the combination of ML210 and olaparib had significant toxic side effects in mice. Figure 6 B).

Claims

1. The application of a GPX4 inhibitor combined with a PARP inhibitor in the preparation of a drug for treating BRCA1-mutated ovarian cancer, wherein the GPX4 inhibitor is ML210, the PARP inhibitor is olaparib, and the combination is a combination of ML210 and olaparib in a dosage ratio of 3:

10.

2. The use of a GPX4 inhibitor in the preparation of a sensitizing agent for a PARP inhibitor against BRCA1-mutated ovarian cancer, wherein the GPX4 inhibitor is ML210, the PARP inhibitor is olaparib, the sensitization is the sensitization of olaparib by ML210, and the dosage ratio of ML210 to olaparib is 3:

10.

3. The application according to claim 1 or 2, characterized in that, The GPX4 inhibitor reverses the tolerance to ferroptosis in BRCA1-mutated ovarian cancer.

4. The application according to claim 3, characterized in that, The BRCA1 enhances the sensitivity of ovarian cancer to ferroptosis by polyubiquitination of GPX4.

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