Application of PLK4-targeted drugs in the treatment of platinum-resistant tumors

Abstract

This article describes the application of PLK4-targeted drugs in the treatment of platinum-resistant tumors. Specifically, it describes the use of PLK4-targeted drugs in the preparation of medicaments for the treatment of oxaliplatin-resistant cancers.

Description

Technical Field

[0001] This application relates to the field of oncology drug therapy, specifically to the application of PLK4-targeted drugs in the treatment of platinum-resistant tumors. Background Technology

[0002] Despite rapid advances in targeted and immunotherapies, chemotherapy, which uses cytotoxic chemotherapeutic agents to disrupt rapidly growing cells, continues to play a crucial role in cancer treatment (Chabner and Roberts, 2005; DeVita and Rosenberg, 2012). Based on their mechanism of action or chemical structure, chemotherapeutic agents can be categorized into different classes, including alkylating agents (which induce DNA damage, such as cisplatin and oxaliplatin), antimetabolites (such as 5-fluorouracil and capecitabine), antitumor antibiotics (DNA intercalators that prevent cell proliferation, such as doxorubicin, also known as doxorubicin), topoisomerase inhibitors (such as irinotecan), and mitotic inhibitors (such as microtubule inhibitors like taxanes, such as docetaxel and paclitaxel) (Huitema et al., 2000; Jiang et al., 2006; Liang et al., 2019; Takeuchi, 1995; Tiwari, 2012). Chemotherapy remains a first-line treatment for many types of cancer. For example, the combination of fluoropyrimidine (e.g., 5-fluorouracil) with oxaliplatin or irinotecan (FOLFOX or FOLFIRI) represents a typical first-line treatment for colorectal cancer (Gustavsson et al., 2015). Importantly, for some unresectable or metastatic cases, chemotherapy may be the only treatment option (Adam, 2003; Bozkurt et al., 2017). More commonly, these chemotherapy drugs are used to shrink tumors or eradicate residual cancer cells before or after surgery (Gosavi et al., 2021; Leon-Ferre et al., 2021). Furthermore, treatment outcomes can be improved by sequential or combined use of chemotherapy with other treatment strategies such as targeted therapy and immunotherapy (Sabanathan et al., 2016; Salas-Benito et al., 2021).

[0003] Drug resistance is a major cause of treatment failure and disease relapse in cancer patients (Holohan et al., 2013; Vasan et al., 2019). Several mechanisms of chemotherapeutic drug resistance have been reported, including reduced drug uptake, increased drug efflux, altered drug targets, drug inactivation, altered DNA repair mechanisms, and impaired cell death signaling. This resistance either originates essentially from a resistant clone pre-existing before treatment or is acquired post-treatment. Genetic and epigenetic factors, such as DNA mutations or gene expression in tumor cells, underlie the development of cell-autonomous chemotherapeutic drug resistance (Holohan et al., 2013; Vaidya et al., 2020). Although several individual genes have been reported to be associated with chemotherapeutic drug resistance, a systematic and comprehensive understanding of the molecular basis behind this phenomenon is urgently needed. These efforts will also facilitate the discovery of genetic biomarkers that can help inform the potential outcomes of chemotherapy and optimize treatment regimens accordingly. More importantly, effective treatment strategies to overcome chemotherapeutic resistance remain limited, significantly restricting the clinical benefits of chemotherapy.

[0004] Colorectal cancer is one of the most common malignant tumors. Many factors can contribute to its development, including genetic and environmental factors (Mariann Bienz, et al., Cell, 2000). Chemotherapy is a crucial treatment for colorectal cancer, with oxaliplatin, a platinum-based chemotherapy drug, playing a significant role in chemotherapy regimens. Combination therapy regimens containing 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) have become representative clinical treatments for colorectal cancer (Martin A. Graham, et al., Clin Cancer Res, 2000). However, after a period of chemotherapy, patients often develop drug resistance, leading to treatment ineffectiveness. Currently, there are still no effective intervention drugs to combat chemotherapy-resistant colorectal cancer.

[0005] Polo-like kinases (PLKs) are key regulatory molecules in the cell cycle. This family has five members: PLK1, 2, 3, 4, and 5. PLK4 has been identified as a major regulator of centrioleum replication; this protein is located in the centrosome and regulates centrosome replication during the cell cycle (Franz Meitinger, et al., Nature, 2020). Studies have shown that PLK4 is overexpressed in various solid tumors, and this high expression is associated with poor clinical outcomes (Jacqueline M. Mason, et al., Cancer Cell, 2014). However, there are currently no reports in the literature regarding the application of PLK4 and its targeted drugs in chemotherapy resistance in colorectal cancer, particularly oxaliplatin resistance. Summary of the Invention

[0006] To identify molecular targets against chemotherapy resistance in colorectal cancer, the inventors constructed multiple colorectal cancer cell lines resistant to oxaliplatin using human colorectal cancer cell lines with diverse genetic backgrounds. Utilizing high-throughput CRISPR gene knockout screening technology and a drug-grade gene CRISPR library, they specifically identified gene targets from thousands of drug-grade genes whose knockout or inactivation significantly enhanced the killing of drug-resistant cells. Among these, inactivation of the PLK4 gene demonstrated significantly greater cytotoxic activity in all drug-resistant cells compared to non-drug-resistant cells.

[0007] This application provides the use of a PLK4-targeted drug in the preparation of a medicament for treating platinum-resistant cancers.

[0008] This application also provides a method for treating platinum-resistant cancer, the method comprising administering a PLK4-targeting drug to a subject in need.

[0009] This application also provides PLK4-targeted drugs for use in the treatment of platinum-resistant cancers.

[0010] In some preferred embodiments, the platinum-resistant cancers are colorectal cancer, lung cancer, breast cancer, ovarian cancer, osteosarcoma, neuroblastoma, cervical cancer, endometrial cancer, vulvar cancer, vaginal cancer, fibrosarcoma, prostate cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, esophageal cancer, chronic myeloid leukemia, gastrointestinal sarcoma, glioblastoma multiforme, fibrous histiocytoma, round cell sarcoma, synovial sarcoma, oropharyngeal cancer, lymphoma, sarcoma, brain cancer, paraneoplastic tumors, anogenital cancer, germ cell tumors, gliomas, or malignant mesothelioma.

[0011] In some preferred embodiments, the platinum-resistant cancer is colorectal cancer.

[0012] In some preferred embodiments, the platinum-based drugs are selected from oxaliplatin, cisplatin, carboplatin, nedaplatin, epplatin, levoplatin, cycloplatin, lobaplatin, ceterplatin, and oxalate platinum.

[0013] In some preferred embodiments, the platinum-based drug is oxaliplatin.

[0014] In some preferred embodiments, the PLK4-targeting drug is a PLK4 inhibitor or a gene-editing drug.

[0015] In some preferred embodiments, the PLK4 inhibitor is selected from CFI-400945, Centrinone (LCR-263), Centrinone-B (LCR-323), CFI-400437, (1E)-CFI-400437 dihydrochloride, PLK4-IN-3, PLK4-IN-1, YLT-11, YLZ-F5, (E)-4-(3-arylvinyl-1H-indazol-6-yl)pyrimidin-2-amine and (E)-3-((1H-indazol-6-yl)methylene)indol-2-one, or pharmaceutically acceptable salts and crystal forms thereof; most preferably, the PLK4 inhibitor is CFI-400945 or a pharmaceutically acceptable salt and crystal form thereof.

[0016] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the solutions described in the description and the accompanying drawings. Attached Figure Description

[0017] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0018] Figure 1 Oxaliplatin-resistant cell lines constructed by knocking out drug resistance-related genes;

[0019] Figure 2 Acquired oxaliplatin-resistant cell lines of colon cancer cells from different genetic backgrounds;

[0020] Figure 3 The HCT116 oxaliplatin-resistant cell lines are sensitive to PLK4 inactivation;

[0021] Figure 4 The DLD1, HCT8, and HT29 oxaliplatin-resistant cell lines are sensitive to PLK4 inactivation;

[0022] Figure 5Knocking out the PLK4 gene in oxaliplatin-resistant cells more significantly inhibits cell division.

[0023] Figure 6 PLK4 gene knockout leads to spindle collapse in oxaliplatin-resistant cells. Blue: DAPI, Green: α-tubulin.

[0024] Figure 7 To demonstrate that each oxaliplatin-resistant HCT116 cell line is sensitive to CFI-400945;

[0025] Figure 8 The cell morphology showed that each oxaliplatin-resistant HCT116 cell line was sensitive to CFI-400945;

[0026] Figure 9 The DLD1, HCT8, and HT29 oxaliplatin-resistant cell lines were made sensitive to CFI-400945.

[0027] Figure 10 Treatment with CFI-400945 resulted in more significant cell cycle arrest in oxaliplatin-resistant cells;

[0028] Figure 11 To show that CFI-400945 treatment caused spindle collapse in oxaliplatin-resistant cells, blue: DAPI, green: α-tubulin;

[0029] Figure 12 In a mouse xenograft model, treatment with CFI-400945 resulted in a more significant reduction in the volume and weight of oxaliplatin-resistant tumors.

[0030] Figure 13 In a mouse xenograft model, treatment with high concentrations of CFI-400945 resulted in a more significant reduction in the volume and weight of tumors formed from acquired oxaliplatin-resistant cells. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, which are not intended to limit the invention in any way.

[0032] This application provides a tumor sample-specific inhibitor that is resistant to platinum-based drugs and combination drugs containing platinum-based drugs, and its application in the treatment of drug-resistant tumors associated with colorectal cancer.

[0033] The inventors first constructed multiple colorectal cancer cell lines with different genetic backgrounds that were resistant to oxaliplatin. By comparing these lines with normal, non-resistant cancer cells, they found that knocking out the PLK4 gene in resistant cells significantly reduced the killing effect on these cells, indicating that resistant cells were more sensitive to PLK4 knockout. PLK4 gene knockout also significantly inhibited cell division in resistant cells.

[0034] Based on this, the inventors used the PLK4-specific inhibitor, compound CFI-400945, or genetic methods such as CRISPR to inactivate the PLK4 gene, as a candidate drug for treating drug resistance associated with colorectal cancer. We found that compound CFI-400945 or CRISPR knockout of PLK4, compared to non-resistant cells, exhibited more significant tumor cell killing activity in various oxaliplatin-resistant colorectal cancer cells; CFI-400945 treatment caused more significant cell cycle arrest in drug-resistant cells; and CFI-400945 also showed a more significant inhibitory effect on oxaliplatin-resistant colorectal cancer tumors in a mouse in vitro xenograft model. Therefore, this invention proposes using the PLK4 inhibitor CFI-400945 as a clinical treatment for colorectal cancer resistant to oxaliplatin and oxaliplatin-containing combination drugs, providing a new and effective treatment and medication strategy for specific drug-resistant patient groups in this situation.

[0035] The inventors proposed using the PLK4-targeted drug CFI-400945 as a clinical treatment for colorectal cancer resistant to oxaliplatin and combination drugs containing oxaliplatin, providing a new and effective treatment and medication strategy for specific drug-resistant patient groups in this situation.

[0036] Therefore, this application provides the use of a PLK4-targeting drug in the preparation of a medicament for treating platinum-resistant cancers.

[0037] This application also provides a method for treating platinum-resistant cancer, the method comprising administering a PLK4-targeting drug to a subject in need.

[0038] This application also provides PLK4-targeted therapies for use in the treatment of platinum-resistant cancers.

[0039] In some embodiments, the PLK4-targeting drug is CFI-400945, Centrinone (LCR-263) (CAS No.: 1798871-30-3), Centrinone-B (LCR-323) (CAS No.: 1798871-31-4), CFI-400437 (CAS No.: 1169211-37-3), (1E)-CFI-400437 dihydrochloride (CAS No.: 1247000-76-5), PLK4-IN-3 (CAS No.: 1247001-86-0), PLK4-IN-1 (CAS No.: 1247001-12-2), YLT-11 (Qian Lei, et al., Cell Death & Disease, 2018) (CAS: N / A), YLZ-F5 (Yongxia Zhu, et al.). (e., Cancer Chemother Pharmacol, 2020)(CAS: N / A), (E)-4-(3-arylvinyl-1H-indazole-6-yl)pyrimidin-2-amine (Zhihao Liu, et al., RSC Advances, 2017)(CAS: N / A), (E)-3-((1H-indazole-6-yl)methylene)indolin-2-ones (Radoslaw Laufer, et al., Journal of Medicinal Chemistry, 2013)(CAS: N / A), etc.

[0040] In some embodiments, the PLK4-targeting drug is a CRISPR agent used to inactivate the PLK4 gene.

[0041] In some preferred embodiments, the PLK4 inhibitor is CFI-400945, or a pharmaceutically acceptable salt and crystal form thereof.

[0042] In some preferred embodiments, CFI-400945 is a compound of formula I disclosed in CN201480064037.9 or a pharmaceutically acceptable salt thereof. In other preferred embodiments, various different crystal forms of CFI-400945 disclosed in CN201480064037.9 may be used.

[0043] In some preferred embodiments, the platinum-based drug is oxaliplatin. Other platinum-based drugs include cisplatin, carboplatin, nedaplatin, epplatin, levoplatin, cycloplatin, lobaplatin, ceterplatin, and oxalate platinum.

[0044] In some preferred embodiments, the platinum-resistant cancer is colorectal cancer.

[0045] Platinum-resistant cancers that can be treated with PLK4 inhibitors include lung cancer, breast cancer, ovarian cancer, osteosarcoma, neuroblastoma, cervical cancer, endometrial cancer, vulvar cancer, vaginal cancer, fibrosarcoma, prostate cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, esophageal cancer, chronic myeloid leukemia, gastrointestinal sarcoma, glioblastoma multiforme, fibrous histiocytoma, round cell sarcoma, synovial sarcoma, oropharyngeal cancer, lymphoma, sarcoma, brain cancer, paraneoplastic tumors, anogenital cancer, germ cell tumors, gliomas, or malignant mesothelioma, etc.

[0046] In some implementations, a PLK4 inhibitor, such as CFI-400945, may be administered to a subject at doses up to about 0.05 mg, 0.1 mg, 0.2 mg, 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times weekly (or no more than three times per week).

[0047] In some implementations, the minimum dose level of the PLK4 inhibitor used to achieve treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg of subject body weight.

[0048] In some implementations, the maximum dose level of the PLK4 inhibitor used to achieve treatment may not exceed approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg of subject body weight.

[0049] In some implementations, the dosage of CFI-400945 is 10-20000 ng / kg subject body weight / day, preferably about 7.5-10 mg / kg subject body weight / day, and more preferably about 10 mg / kg subject body weight / day.

[0050] In some implementations, the PLK4 inhibitor used to achieve the treatment can be administered to the subject via a variety of routes, including but not limited to oral, local, oral, sublingual, pulmonary, transdermal, transmucosal, subcutaneous, intraperitoneal, intravenous, and intramuscular injection, or administration in liquid or solid doses via the digestive tract.

[0051] In some embodiments, the PLK4 inhibitor for achieving treatment may be formulated as a pharmaceutical composition in a solid dosage form. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form may be, for example, an instant-dissolving form, a controlled-release form, a lyophilized form, a delayed-release form, an extended-release form, a pulsatile-release form, a mixture of immediate-release and controlled-release forms, or a combination thereof.

[0052] In some embodiments, PLK4 inhibitors can be formulated into pharmaceutical compositions comprising a carrier. For example, the carrier may be selected from proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0053] The pharmaceutical composition may include one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers may include lactose monohydrate, anhydrous lactose, and various starches; examples of binders are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose such as Avicel PH101 and Avicel PH102, and silicified microcrystalline cellulose (ProSolv SMCC). TM Suitable lubricants, including agents that improve the flowability of the powder to be compressed, may include colloidal silica, such as Aerosil 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweeteners, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame potassium. Examples of flavoring agents are Magnasweet (a trademark of MAFCO), bubble gum flavoring, fruit flavorings, etc. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of benzoic acid such as butylparaben, alcohols such as ethanol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0054] Suitable diluents may include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dicalcium phosphate, sugars, and any mixtures thereof. Examples of diluents include microcrystalline cellulose such as Avicel PH101 and Avicel PH102; lactose such as lactose monohydrate, anhydrous lactose, and Pharmatose DCL21; dicalcium phosphate such as Emcompress; mannitol; starch; sorbitol; sucrose; and glucose.

[0055] Suitable disintegrants include mildly cross-linked polyvinylpyrrolidone, corn starch, potato starch, corn starch and modified starch, croscarmellose sodium, croscarmellose, glycolic acid starch sodium and mixtures thereof.

[0056] Examples of effervescent agents are effervescent pairs, such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, and alginic acid, as well as acid anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent pair may be present.

[0057] In some embodiments, the PLK4 inhibitor for achieving the treatment can be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition can be administered orally, intravenously, intramuscularly, subcutaneously, or topically. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders, and granules.

[0058] In some embodiments, PLK4 inhibitors for achieving treatment can be administered in conventional dosage forms prepared by combining the active ingredient with a standard drug carrier or diluent according to standard procedures known in the art. These procedures may involve mixing, granulating, and compressing or dissolving the ingredients to suit the desired formulation.

[0059] Pharmaceutical compositions containing PLK4 inhibitors are suitable for administration via any suitable route, such as oral (including oral or sublingual), rectal, nasal, local (including oral, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal and intrathecal) routes. Such pharmaceutical compositions can be prepared by any method known in the pharmaceutical field, such as by conjugating the active ingredient with a carrier or excipient.

[0060] Pharmaceutical compositions suitable for oral administration may exist in the form of discrete units, such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; or oil-in-water or water-in-oil liquid emulsions.

[0061] Tablets and capsules for oral administration may be in unit dose form and may contain conventional excipients, such as binders like syrup, gum arabic, gelatin, sorbitol, tragali gum, or polyvinylpyrrolidone; fillers like lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; compression lubricants like magnesium stearate, talc, polyethylene glycol, or silica; disintegrants like potato starch; or acceptable wetting agents like sodium lauryl sulfate. Tablets may be coated according to methods well-known in conventional pharmaceutical practice. Oral liquid formulations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be reconstituted with water or other suitable carriers before use as a dried product. Such liquid formulations may contain conventional additives, such as suspending agents, such as sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifiers, such as lecithin, dehydrated sorbitol monooleate, or gum arabic; non-aqueous carriers (which may include edible oils, such as almond oil); oily esters such as glycerin, propylene glycol or ethanol; preservatives, such as methylparaben or propylparaben or sorbic acid; and, if desired, conventional flavorings or colorings.

[0062] The following provides specific materials used in embodiments of the present invention and their sources. However, it should be understood that these are merely exemplary and are not intended to limit the invention.

[0063] The cell biology experimental techniques used in the implementation include cell culture, cell transfection, cell infection, and cell counting. Mouse experimental techniques include subcutaneous tumor implantation and gavage in mice. Unless otherwise specified, the procedures are usually performed according to standard methods.

[0064] abbreviation:

[0065] HCT116-Mock consists of untreated HCT116 cells;

[0066] HCT116-Vector is HCT116 cells infected with the lentiCRISPR-v2 vector;

[0067] HCT116-AAVS1 is HCT116 cells that have had AAVS1 knocked out using the CRISPR / Cas9 system. AAVS1 is currently defined as a safe harbor site, meaning that knocking it out has no significant functional impact on the genome.

[0068] HCT116-ΔTP53 OR -1 and HCT116-ΔTP53 OR-2 is a drug-resistant cell line constructed by knocking out TP53 in HCT116 using the CRISPR / Cas9 system. "Δ" represents knockout, and OR is short for oxaliplatin resistance. Because two pairs of sgRNAs were designed for the gene, they were named -1 and -2.

[0069] HCT116-ΔSLC43A2 OR -1 and HCT116-ΔSLC43A2 OR -2 is a drug-resistant cell line constructed by knocking out SLC43A2 in HCT116 using the CRISPR / Cas9 system. "Δ" represents knockout, and OR is short for oxaliplatin resistance. Because two pairs of sgRNAs were designed for the gene, they were named -1 and -2.

[0070] HCT116-ΔCDKN1A OR -1 and HCT116-ΔCDKN1A OR -2 is a drug-resistant cell line constructed by knocking out CDKN1A in HCT116 using the CRISPR / Cas9 system. "Δ" represents knockout, and OR is short for oxaliplatin resistance. Because two pairs of sgRNAs were designed for the gene, they were named -1 and -2.

[0071] HCT116 OR DLD1 OR HCT8 OR and HT29 OR These are acquired oxaliplatin-resistant strains constructed by gradually increasing the oxaliplatin drug concentration from various wild-type colon cancer cells.

[0072] First, multiple oxaliplatin-resistant colon cancer cell lines with different genetic backgrounds were constructed. The oxaliplatin-resistant cell line HCT116-ΔTP53 was constructed from gene knockout cells. OR HCT116-ΔSLC43A2 OR and HCT116-ΔCDKN1A OR ( Figure 1 (as shown) and the acquired resistant strain HCT116 constructed by progressively increasing oxaliplatin concentrations from normal colon cells. OR DLD1 OR HCT8 OR and HT29 OR ( Figure 2 (As shown).

[0073] By comparing with control non-drug-resistant cancer cells such as HCT116-Mock, HCT116-Vector, and HCT116-AAVS1, we found that knocking out the PLK4 gene in drug-resistant cells significantly killed drug-resistant cells, meaning that drug-resistant cells are more sensitive to PLK4 knockout. Figure 3 and Figure 4 (As shown).

[0074] The PLK4 protein is located in the centrosome and regulates centrosome replication during the cell cycle. We found that, compared to normal non-drug-resistant cancer cells, PLK4 gene knockout significantly inhibited cell division in drug-resistant cells. Figure 5 (As shown). Cells with PLK4 knockout formed short bipolar spindles. Compared to the non-resistant Vector group, oxaliplatin-resistant cells exhibited spindle collapse, arresting or slipping out of mitosis without undergoing anaphase, leading to chromosome segregation failure and ultimately growth arrest. Figure 6 (As shown).

[0075] Based on the above experimental results, we selected the PLK4-specific inhibitor, compound CFI-400945, as a candidate drug for treating colorectal cancer-related drug resistance. We found that compound CFI-400945, compared to non-resistant cells, exhibited significantly more tumor cell killing activity in various oxaliplatin-resistant colorectal cancer cells. Figure 7 and Figure 9 As shown in the figure). In terms of cell morphology, it is clearly evident that, compared to non-resistant cells, oxaliplatin-resistant cell lines are sensitive to CFI-400945, exhibiting significant morphological deterioration and membrane fusion. Figure 8 As shown). CFI-400945 treatment caused more significant cell cycle arrest in drug-resistant cells (as shown). Figure 10 (As shown). Cells treated with CFI-400945 formed short bipolar spindles. Compared to the non-resistant Vector group, oxaliplatin-resistant cells exhibited spindle collapse, arresting or slipping out of mitosis without undergoing anaphase, leading to chromosome separation failure and ultimately growth arrest. Figure 11 (As shown).

[0076] In a mouse xenograft model, CFI-400945 also showed a more significant inhibitory effect on oxaliplatin-resistant colorectal cancer tumors. The mouse experiment was divided into six groups: subcutaneous tumor implantation with non-resistant cells and oxaliplatin-resistant cells. Six days after subcutaneous tumor implantation, HCT116-Vector and HCT116-ΔSLC43A2 cells were treated with vehicle and CFI-400945, respectively. OR and HCT116OR Mice in the experimental group were administered CFI-400945 via gavage at a concentration of 7.5 mg / kg / day. At the experimental endpoint, compared with HCT116-Vector-CFI-400945, HCT116-ΔSLC43A2... OR -CFI-400945 and HCT116 OR The tumor in CFI-400945 showed a significant reduction in both volume and weight. Figure 12 (As shown).

[0077] based on Figure 12 In our experiment, we readjusted the dosage of CFI-400945 to 10 mg / kg / day. At this dosage, at the experimental endpoint, compared to HCT116-Vector-CFI-400945, HCT116... OR- The tumor in CFI-400945 showed a more significant reduction in both volume and weight. Figure 13 (As shown).

[0078] In the following embodiments:

[0079] Human HCT116 (Cat#CCL-247), DLD1 (Cat#CCL-221), HCT8 (Cat#CCL-244), and HT29 (Cat#HTB-38) cells were obtained from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO2 in DMEM medium (HCT116 and HT29 cells) or RPMI 1640 medium (DLD1 and HCT8 cells) containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0080] DMEM was purchased from BI, Cat#06-1055-57-1ACS. RPMI-1640 was purchased from BI, Cat#01-100-1ACS.

[0081] Oxaliplatin was purchased from the National Institutes for Food and Drug Control (Cat#100584), CFI-400945 (CAS#1616420-30-4) was purchased from MCE (Cat#HY-12300B), nude mice used in the examples were purchased from Beijing Huafukang Biotechnology Co., Ltd., and the sgRNA sequences used in the examples are shown in Table 1 and were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0082] Table 1. sgRNA target sequences

[0083]

[0084]

[0085] Example 1: Oxaliplatin-resistant cell line constructed by knocking out drug resistance-related genes

[0086] Gene knockout was performed using the CRISPR-Cas9 system. Two sgRNAs were designed for each gene, and oxaliplatin-resistant cell lines were constructed by knocking out genes using sgRNAs. HCT116 cell lines with each gene knockout resulted in a significant resistant cell population after treatment with 5 μM oxaliplatin for 6 days. This indicates that mutations or inactivation of TP53, SLC43A2, and CDKN1A can lead to a chemotherapy-resistant phenotype. IC50 was calculated. 50 The cell viability assay is based on the MTT colorimetric method. Succinate dehydrogenase in the mitochondria of live cells can reduce exogenous MTT to water-insoluble blue-violet formazan crystals, which are then deposited within the cell. Dead cells lack this function. The number of MTT crystals formed is directly proportional to the cell number. The number of live cells is then measured based on absorbance values. Cells (4 x 10⁶ cells per well) are then used. 3 Cells were seeded in 96-well plates and, after adhesion, treated with oxaliplatin dissolved in DMSO for 3 days. Three replicates were set up for each drug concentration, with an equal volume of DMSO added as a blank control. Then, 10 μL of MTT (5 mg / mL) was added to the upper part of each well, gently shaken, and incubated for 4 hours. Afterward, the culture medium was gently aspirated, and 100 μL of DMSO was added to each well, gently shaken for 5 minutes to promote complete dissolution of crystals. Cell viability was determined using a BioTek spectrophotometer (Gene Company Limited) at 490 nm absorbance, and IC50 was calculated using Prism9 software. 50 value. Figure 1 The top figure shows the cell growth curves of HCT116 knockout gene resistant cell lines under different concentrations of oxaliplatin treatment, as determined by the MTT assay. The bottom figure shows the IC50 values ​​of oxaliplatin for HCT116 knockout gene resistant cell lines. 50 A bar chart of values.

[0087] Example 2: Acquired oxaliplatin-resistant cell lines of colon cancer cells with different genetic backgrounds

[0088] Using colon cancer cells from four different genetic backgrounds—HCT116, DLD1, HCT8, and HT29—the HCT116 strain was constructed by gradually increasing the concentration of oxaliplatin (starting from 1 μM and gradually increasing the concentration) after approximately 2-3 months. OR DLD1 OR HCT8 OR and HT29OR The final oxaliplatin treatment concentrations for HCT116, DLD1, HCT8, and HT29 cells were 45 μM, 80 μM, 50 μM, and 35 μM, respectively. Approximately one week after treatment at these final concentrations, significant cell death was observed in the corresponding control groups of normal HCT116, DLD1, HCT8, and HT29 cells, clearly demonstrating that all four cell lines successfully developed acquired drug resistance. Figure 2 "**" indicates that compared with the non-drug-resistant control group, p<0.01.

[0089] Example 3: HCT116 oxaliplatin-resistant cell lines are sensitive to PLK4 inactivation.

[0090] Ten cell lines, HCT116-Mock, HCT116-Vector, HCT116-AAVS1, and HCT116-ΔTP53, were used. OR -1、HCT116-ΔTP53 OR -2、HCT116-ΔSLC43A2 OR -1、HCT116-ΔSLC43A2 OR -2、HCT116-ΔCDKN1A OR -1、HCT116-ΔCDKN1A OR -2 and HCT116 OR The study was divided into a control group (sgCtrl: an empty vector without sgRNA) and an experimental group (sgPLK4-1, sgPLK4-2: two sgRNAs of PLK4 knocked out by CRISPR; sgPLK1-1, sgPLK1-2: two sgRNAs of PLK1 knocked out by CRISPR).

[0091] HCT116 cells were lysed for 15 minutes at 4°C in RIPA buffer (Beyotime) containing a phosphatase inhibitor (Meilunbio#MB12707) and a protease inhibitor (Meilunbio#MB26780). Afterwards, the cells were centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The protein supernatant was mixed with loading buffer, and the proteins were separated on a 10% bis-tris polyacrylamide gel. The proteins in the gel were transferred to a nitrocellulose (NC) membrane (PallCorporation#27574625) and blocked with 5% skim milk (Difco™ Skim Milk#4296916). After incubation with primary antibody (PLK4; Cell Signaling Technology; Cat#71033 and PLK1; Proteintech; Cat#10305-1-AP) and secondary antibody (Goat anti-Rabbit IgG; Thermo Fisher Scientific; Cat#31460), the expression of PLK4 and PLK1 proteins was detected using a chemiluminescence imaging system (Tanon-5200). Infection with the corresponding CRISPR sgRNA virus successfully reduced the protein expression levels of either PLK4 or PLK1 in HCT116 cells. Figure 3 superior).

[0092] Eleven days after infection with the corresponding sgRNA virus, cell counts were performed using a hemocytometer on the above 10 cell lines. When PLK4 was knocked out, the number of surviving cells was significantly lower in all tested drug-resistant cells than in non-drug-resistant cells, indicating that drug-resistant cell lines are more sensitive to PLK4 inactivation. Figure 3 (in Chinese); while knocking out PLK1 only had a weak killing effect on a few drug-resistant cells. Figure 3 (See below) This indicates that PLK4 is a more suitable molecular target for combating drug resistance than PLK1. "**" indicates that compared with Mock, Vector, and AAVS1 within each group, p < 0.01.

[0093] Example 4: DLD1, HCT8, and HT29 oxaliplatin-resistant cell lines are sensitive to PLK4 inactivation.

[0094] DLD1, HCT8, and HT29 oxaliplatin-resistant cell lines were divided into a control group (sgCtrl: an empty vector without sgRNA) and an experimental group (sgPLK4-1, sgPLK4-2: two sgRNAs with PLK4 knocked out by CRISPR). Cell growth was observed after infection with the corresponding sgRNA viruses, and cell counts were performed using a hemocytometer. After PLK4 knockout, the number of surviving resistant cells was significantly lower than that of non-resistant cells. In other words, similar to the conclusions obtained in Example 3, DLD1, HCT8, and HT29 resistant cell lines are also more sensitive to PLK4 inactivation. Figure 4 "*" indicates p < 0.05 compared to the non-drug-resistant control group; "**" indicates p < 0.01 compared to the non-drug-resistant control group.

[0095] Example 5: PLK4 gene knockout significantly inhibits cell division in drug-resistant cells.

[0096] In this embodiment, four cell lines of HCT116 (Vector, ΔSLC43A2, etc.) were used. OR ΔTP53 OR HCT116 ORCells were divided into a control group (sgAAVS1) and an experimental group (sgPLK4-1). Twelve days after infection, each treatment group was treated with 75 μM 5-bromodeoxyuridine (BrdU) for 1 h. Cells were thoroughly digested and washed with PBS. 90% ethanol was added dropwise under vortex conditions to fix the cells. The cells were incubated overnight at 4°C in the dark. Samples were resuspended and washed with PBS. 0.5 mL of 2M HCl (0.5% Triton X-100) was added, and the cells were vortexed and incubated for 30 min at room temperature, followed by washing with PBS. 1 mL of 100 mM sodium borate solution (pH = 8.5) was added to resuspend the cells, and the cells were centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and 1 mL of 3% BSA blocking solution (PBS-0.5% Tween 20, PBST) was added. The cells were incubated for 30 min at room temperature, followed by centrifugation to remove the supernatant. Add 100 μL of BrdU primary antibody (Cell Signaling Technology #5292; 1:200) and incubate at room temperature for one hour. Wash with PBST, then add 100 μL of mouse fluorescent secondary antibody (Thermo Fisher Scientific #A-11001; 1:500). Incubate in the dark at room temperature for 30 minutes, then wash with PBST. Add 350 μL of PI / RNase Staining Buffer (BD #550825) and incubate at room temperature in the dark for 30 minutes. Filter the sample through a 200-mesh nylon membrane. Run the sample on a flow cytometer (BD LSR Fortessa) and analyze the cell cycle distribution using FlowJo_v10 software. After PLK4 knockout, compared with the non-resistant cell group, the cell cycle of each drug-resistant cell was significantly arrested at the G2 / M phase, delaying the mitotic process. Figure 5 ).

[0097] Example 6: PLK4 gene knockout leads to spindle collapse in drug-resistant cells

[0098] In this embodiment, four cell lines of HCT116 (Vector, ΔSLC43A2, etc.) were used. OR ΔTP53 OR HCT116 ORCells were divided into a control group (sgAAVS1) and an experimental group (sgPLK4-1). Ten days after infection, cells were seeded in 24-well plates with circular slides. After cell adhesion, cells were treated with 2 mM thymidine for 16 hours, followed by 8 hours of culture in normal medium. This process was repeated once, treating cells with 100 ng / mL nocodazole for 5 hours to complete cell synchronization. Cells were then fixed at five time points: 0 min, 30 min, 45 min, 60 min, and 90 min. Before fixation, cells were washed three times with PBS for 3 minutes each time. Slides were then fixed with 3.7% paraformaldehyde for 10 minutes, followed by three washes with PBS for 3 minutes each time. Cell membranes were permeabilized with 0.1% Triton X-100 (prepared with PBS) at room temperature for 2 minutes, followed by three washes with PBS. Slides were then blocked with bovine serum albumin (BSA) at room temperature for 30 minutes. Diluted primary antibody (α-tubulin 1:100, Proteintech #66031-1-Ig) was added to each slide. The slides were placed in a humidified chamber and incubated overnight at 4°C. The slides were then washed three times with PBST for 3 minutes each time, followed by incubation with fluorescent secondary antibody (goat anti-mouse IgG, 1:500, Thermo Fisher Scientific #A-11001) at room temperature for 1 hour. The slides were then washed three times with PBST for 3 minutes each time. DAPI was added and incubated for 5 minutes, followed by three washes with PBST for 5 minutes each time. The slides were dried with absorbent paper and sealed with an anti-fluorescence quencher. Images were collected using a confocal microscope (Leica TCS SP8). After PLK4 knockout, compared to the non-resistant cell group, all drug-resistant cells exhibited spindle collapse, folding, and slippage. Figure 6 This causes chromosome separation to fail, ultimately leading to growth arrest.

[0099] Example 7: HCT116 oxaliplatin-resistant cell lines sensitive to CFI-400945

[0100] The cell lines used in this example are the same as in Example 3. Cell counting was performed using a hemocytometer, and the same conclusion was reached as in Example 3: the drug-resistant cell lines are more sensitive to the PLK4 inhibitor CFI-400945. In other words, CFI-400945 can more effectively kill oxaliplatin-resistant cell lines. Figure 7 This example uses CFI-400945 at a concentration of 12 nM. "**" indicates p < 0.01 compared to Mock, Vector, and AAVS1 within each group.

[0101] Example 8: Cell morphology showed that each oxaliplatin-resistant HCT116 cell line was sensitive to CFI-400945.

[0102] In this example, the ten cell lines were plated with the same amount as in Example 3. After cell adhesion, the cells were treated with 12 nM CFI-400945 for 7 days. Compared with non-resistant cell lines, the PLK4 inhibitor CFI-400945 had a stronger killing effect on resistant cell lines, and the cells showed morphological deterioration and membrane fusion. Figure 8 ).

[0103] Example 9: DLD1, HCT8 and HT29 oxaliplatin-resistant cell lines sensitive to CFI-400945

[0104] DLD1, HCT8, and HT29 oxaliplatin-resistant and non-resistant cell lines were treated with CFI-400945 at concentrations of 10 nM, 15 nM, and 15 nM, respectively. Cell counts were performed using a hemocytometer, yielding conclusions similar to those in Example 7, namely, that resistant cell lines are more sensitive to the PLK4 inhibitor CFI-400945, or in other words, CFI-400945 can more effectively kill oxaliplatin-resistant cell lines. Figure 9 "*" indicates p < 0.05 compared to the non-drug-resistant control group; "**" indicates p < 0.01 compared to the non-drug-resistant control group.

[0105] Example 10: CFI-400945 treatment resulted in more significant cell cycle arrest in oxaliplatin-resistant cells.

[0106] The four cell lines HCT116 in this embodiment OR HCT116-ΔTP53 OR HCT116-ΔSLC43A2 OR HCT116-Vector cells were treated with DMSO and 12 nM CFI-400945 for 7 days, respectively. Specific experimental procedures are detailed in Example 5. Compared to the non-resistant cell group, CFI-400945 treatment significantly arrested the cell cycle of all resistant cells at the G2 / M phase, delaying the progression of mitosis. Figure 10 ).

[0107] Example 11: Treatment with CFI-400945 led to spindle collapse in drug-resistant cells.

[0108] The four cell lines HCT116 in this embodiment OR HCT116-ΔTP53 OR HCT116-ΔSLC43A2 ORHCT116-Vector cells were treated with DMSO and 12 nM CFI-400945 for 7 days, respectively. Specific experimental procedures are detailed in Example 6. After CFI-400945 treatment, compared to the non-resistant cell group, all resistant cells exhibited spindle collapse, folding, and slippage, leading to chromosome segregation failure and ultimately growth arrest. Figure 11 ).

[0109] Example 12: In a mouse xenograft model, oxaliplatin-resistant tumors treated with CFI-400945 showed a more significant reduction in volume and weight.

[0110] The mouse experiment was divided into six experimental groups: the non-oxaliplatin-resistant cell line HCT116-Vector, and the oxaliplatin-resistant cell line HCT116-ΔSLC43A2. OR and HCT116 OR , respectively with 3*10 6 One cell was used to subcutaneously implant tumors in mice on both sides of the skin. When the tumor volume was approximately 100 mm... 3 At time (6 days post-tumor implantation), HCT116-Vector and HCT116-ΔSLC43A2 were compared with solvent control (DMSO) and CFI-400945, respectively. OR and HCT116 OR Mice in the experimental group were administered CFI-400945 via gavage at a concentration of 7.5 mg / kg / day, 5 days per week. At the experimental endpoint (30 days after tumor implantation), the tumors were dissected, and their volume and weight were measured. The tumor volume was calculated using the following formula: Volume = (Length * Width * Width) / 2. Compared to the non-resistant cell group, the CFI-400945 treatment significantly reduced the tumor volume and weight in all resistant cell groups. Figure 12 Among them, HCT116-Vector group, HCT116-ΔSLC43A2 OR and HCT116 OR The tumor volume inhibition rates were 36.9%, 54.5%, and 42%, respectively, and the tumor weight inhibition rates were 22.5%, 55.2%, and 36.4%, respectively. Tumor volume inhibition rate = (average volume of control group - average volume of experimental group) / average volume of control group × 100%. Tumor weight inhibition rate = (average mass of control group - average mass of experimental group) / average mass of control group × 100%. n = 17 (Vector-Vehicle), n = 18 (Vector-CFI-400945), n = 15 (ΔSLC43A2) OR -1-Vehicle), n=15 (ΔSLC43A2 OR -1-CFI-40094), n=17(HCT116)OR -Vehicle), n=17(HCT116) OR -CFI-400945). "*" indicates p<0.05 compared to the control group; "**" indicates p<0.01 compared to the control group.

[0111] Example 13: In a mouse xenograft model, treatment with high concentrations of CFI-400945 resulted in a more significant reduction in the volume and weight of tumors formed by acquired oxaliplatin-resistant cells.

[0112] The mouse experiment was divided into four experimental groups: the non-oxaliplatin-resistant cell line HCT116-Vector, the oxaliplatin-resistant cell line HCT116... OR , respectively with 3*10 6 One cell was used to subcutaneously implant tumors in mice on both sides of the skin. When the tumor volume was approximately 100 mm², 3 At time (6 days post-tumor implantation), HCT116-Vector and HCT116 were compared with solvent control (DMSO) and CFI-400945, respectively. OR Mice in the experimental group were administered CFI-400945 via gavage at a concentration of 10 mg / kg / day, 5 days a week. At the experimental endpoint (22 days after tumor implantation), the tumors were dissected, and their volume and weight were measured. The tumor volume was calculated using the following formula: Volume = (Length * Width * Width) / 2. Compared to the non-resistant cell group, the CFI-400945 treatment significantly reduced the tumor volume and weight in the resistant cell group. Figure 13 Among them, the HCT116-Vector group and HCT116 OR The tumor volume inhibition rates were 25.6% and 83.7%, respectively, and the tumor weight inhibition rates were 57.3% and 79%, respectively. Compared to Example 12 ( Figure 12 HCT116 OR The tumor volume inhibition rate was 42% and the tumor weight inhibition rate was 36.4%, with the CFI-400945 drug concentration of 10 mg / kg / day showing more significant effects. Tumor volume inhibition rate = (average volume of control group - average volume of experimental group) / average volume of control group × 100%. Tumor weight inhibition rate = (average mass of control group - average mass of experimental group) / average mass of control group × 100%. n = 4 (Vector-Vehicle), n = 5 (Vector-CFI-400945), n = 5 (HCT116) OR -Vehicle), n=5(HCT116) OR-CFI-400945). "*" indicates p<0.05 compared to the control group; "**" indicates p<0.01 compared to the control group; "****" indicates p<0.0001 compared to the control group.

[0113] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

Claims

1. The use of PLK4-targeted drugs in the preparation of drugs for the treatment of platinum-resistant colorectal cancer, wherein, The PLK4-targeting drug is CFI-400945, or its pharmaceutically acceptable salts and crystal forms.

2. The use according to claim 1, wherein the platinum-based drug is selected from oxaliplatin, cisplatin, carboplatin, nedaplatin, epplatin, levoplatin, cycloplatin, lobaplatin, ceterplatin, and oxalate platinum.

3. The use according to claim 2, wherein the platinum-based drug is oxaliplatin.

4. The use according to claim 1, wherein, The dosage of CFI-400945 is 7.5-10 mg / kg subject body weight / day.

5. The use according to claim 1, wherein, The dosage of CFI-400945 is 10 mg / kg of subject body weight / day.

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