CN116850289A

CN116850289A - Application of PLK4 targeted drug in treatment of platinum drug resistant tumor

Info

Publication number: CN116850289A
Application number: CN202310825368.0A
Authority: CN
Inventors: 费腾; 钟春鸽; 李泽旭
Original assignee: 东北大学
Priority date: 2022-07-29
Filing date: 2023-07-06
Publication date: 2023-10-10
Also published as: WO2024022080A1

Abstract

Provided herein are uses of PLK4 targeted drugs in the treatment of tumors resistant to platinum drugs. In particular, provided herein is the use of PLK4 targeted drugs in the manufacture of a medicament for the treatment of oxaliplatin-resistant cancers.

Description

Application of PLK4 targeted drug in treatment of platinum drug resistant tumor

Technical Field

The application relates to the field of tumor drug treatment, in particular to application of PLK4 targeted drugs in treating platinum drug resistant tumors.

Background

Despite rapid advances in targeted and immunotherapy, chemotherapy using cytotoxic chemicals to destroy rapidly growing cells still plays a critical role in cancer therapy (Chabner and Roberts,2005;DeVita and Rosenberg,2012). Depending on the mechanism of action or chemical structure, chemical drugs can be divided into different classes, including alkylating agents (inducing DNA damage, e.g. cisplatin and oxaliplatin), antimetabolites (e.g. 5-fluorouracil and capecitabine), antitumor antibiotics (DNA intercalators that prevent cell proliferation, e.g. doxorubicin, also known as doxorubicin), topoisomerase inhibitors (e.g. irinotecan) and mitotic inhibitors (e.g. microtubule inhibitor taxanes, e.g. docetaxel and paclitaxel) (Huitema et al, 2000; jiang et al, 2006; liang et al, 2019; takeuchi,1995; tiwari, 2012). Chemotherapy remains the first line treatment for many types of cancer. For example, the use of the chemical fluoropyrimidine (e.g., 5-fluorouracil) in combination with oxaliplatin or irinotecan (FOLFOX or FOLFIRI) represents a typical first-line treatment of colorectal cancer (gustawsson et al, 2015). Importantly, for some unresectable or metastatic cases, chemotherapeutic drugs may be the only treatment regimen (Adam, 2003;Bozkurt et al, 2017). More commonly, these chemotherapeutics are used to shrink tumors or eradicate residual cancer cells before and after surgery (Gosavi et al, 2021; leon-Ferre et al, 2021). Furthermore, therapeutic effects can be enhanced by sequential or combined application of chemotherapy and other therapeutic strategies such as targeted therapy and immunotherapy (Sabanathan et al 2016; salas-Benito et al 2021).

Drug resistance is often the primary cause of treatment failure and disease recurrence in cancer patients (Holohan et al, 2013; vasan et al, 2019). Several mechanisms of chemotherapy drug resistance have been reported, such as decreased drug uptake, increased drug efflux, altered drug targets, drug inactivation, altered DNA repair mechanisms, and impaired cell death signaling. Such resistance is either essentially from pre-treatment existing resistant clones or is obtained after treatment. Genetic and epigenetic factors, such as DNA mutations or gene expression in tumor cells, are the basis for the development of resistance to autonomous chemotherapeutics by cells (Holohan et al, 2013;Vaidya et al, 2020). Although several individual genes have been reported to be involved in chemotherapeutic drug resistance, there is an urgent need for a systematic and comprehensive understanding of the molecular basis behind this phenomenon. These efforts will also facilitate the discovery of gene biomarkers that help inform the potential outcome of chemotherapy and optimize treatment regimens accordingly. More importantly, effective therapeutic strategies to overcome chemotherapy resistance remain limited, which significantly limits the clinical benefit of chemotherapy.

Colorectal cancer is one of the most common malignant tumors. Many factors may lead to the development of this disease, including genetic and environmental factors (Mariann Bienz, et al, cell, 2000). Chemotherapeutic agents are one of the important means for treating colon Cancer, wherein oxaliplatin as a platinum-based chemotherapeutic agent occupies a significant position in the chemotherapeutic regimen of colon Cancer, and a combined therapeutic regimen comprising 5-fluorouracil, folinic acid, oxaliplatin (FOLFOX) and other chemotherapeutic agents has become a representative therapeutic means for clinically treating colon Cancer (Martin a. Graham, et al, clin Cancer Res, 2000). However, after a period of chemotherapy treatment, patients often develop tolerance to the drug, resulting in ineffective treatment. For chemotherapy resistance, there is currently a lack of effective intervention drugs against chemotherapy-resistant colorectal cancer.

Polo-like kinases (PLKs) are key regulatory molecules during the cell cycle. There are five members of this family: PLKs 1, 2, 3, 4 and 5.PLK4 has been identified as a major regulator of centrosome replication, and this protein localizes to the centrosome and regulates centrosomal replication during the cell cycle (Franz Meitinger, et al, nature, 2020). Studies have shown that PLK4 is overexpressed in a variety of solid tumors, and that this high expression is associated with poor clinical outcome (Jacqueline M.Mason, et al, cancer Cell, 2014). However, no document reports about the application of PLK4 and targeted drugs thereof in colorectal cancer chemotherapeutic drug resistance, in particular to the action of oxaliplatin in drug resistance.

Disclosure of Invention

In order to find a molecular target for resisting colorectal cancer chemotherapy drug resistance, the inventor constructs a plurality of colorectal cancer cells with drug resistance to oxaliplatin by utilizing human colorectal cancer cell lines with various different genetic backgrounds, and specifically identifies gene targets of which the gene knockout or inactivation can better kill drug resistant cells from thousands of patent drug genes by utilizing a high-throughput CRISPR gene knockout screening technology and a patent drug gene CRISPR library. Wherein, the functional inactivation of PLK4 gene shows more remarkable cell killing activity in all drug-resistant cells compared with non-drug-resistant cells.

The application provides application of PLK4 targeted drugs in preparing drugs for treating platinum drug-resistant cancers.

The application also provides a method of treating platinum drug resistant cancer comprising administering a PLK4 targeting drug to a subject in need thereof.

The application also provides PLK4 targeted drugs for use in the treatment of platinum drug resistant cancers.

In some preferred embodiments, the platinum drug resistant cancer is 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 granulocytic leukemia, gastrointestinal sarcoma, glioblastoma multiforme, fibrohistiocytoma, round cell tumor sarcoma, synovial sarcoma, oropharyngeal cancer, lymphoma, sarcoma, brain cancer, paratumor, anogenital cancer, germ cell tumor, glioma, or malignant mesothelioma.

In some more preferred embodiments, the platinum drug resistant cancer is colorectal cancer.

In some preferred embodiments, the platinum-based drug is selected from oxaliplatin, cisplatin, carboplatin, nedaplatin, cisplatin, lobaplatin, satraplatin, and platinum oxalate.

In some more preferred embodiments, the platinum-based drug is oxaliplatin.

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

IN some more 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 crystalline forms thereof; most preferably, the PLK4 inhibitor is CFI-400945 or a pharmaceutically acceptable salt and crystalline form thereof.

Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. Other advantages of the application may be realized and attained by the structure particularly pointed out in the written description and drawings.

Drawings

The accompanying drawings are included to provide an understanding of the principles of the application, and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain, without limitation, the principles of the application.

FIG. 1 is an oxaliplatin resistant cell line constructed by knocking out a drug resistance related gene;

FIG. 2 is an acquired oxaliplatin resistant cell line of colon cancer cells of different genetic backgrounds;

FIG. 3 shows that each oxaliplatin-resistant cell line of HCT116 is susceptible to inactivation by PLK4;

FIG. 4 shows that DLD1, HCT8 and HT29 oxaliplatin resistant cell lines are susceptible to inactivation by PLK4;

FIG. 5 shows that PLK4 gene knockout blocks the progression of cell division to a greater extent in oxaliplatin-resistant cells;

fig. 6 shows that PLK4 gene knockout resulted in spindle collapse in oxaliplatin resistant cells, blue: DAPI, green: alpha-tubulin (alpha-tubulin);

FIG. 7 shows that each oxaliplatin-resistant cell line of HCT116 is susceptible to CFI-400945;

FIG. 8 is a graph showing cell morphology that HCT116 individual oxaliplatin resistant cell lines are susceptible to CFI-400945;

FIG. 9 shows that DLD1, HCT8 and HT29 oxaliplatin resistant cell lines are susceptible to CFI-400945;

FIG. 10 shows that oxaliplatin-resistant cells developed a more pronounced cell cycle arrest upon CFI-400945 treatment;

FIG. 11 is a graph showing that CFI-400945 treatment resulted in spindle collapse in oxaliplatin-resistant cells, blue: DAPI, green: alpha-tubulin;

FIG. 12 is a graph showing that oxaliplatin-resistant tumors developed a more pronounced volume and weight reduction in the mouse xenograft model, treated with CFI-400945;

figure 13 is a graph showing that the tumor formation of acquired oxaliplatin resistant cells resulted in a more significant volume and weight reduction in the mouse xenograft model treated with high concentrations of CFI-400945.

Detailed Description

The application is further illustrated by the following examples, which are not intended to be limiting in any way, in conjunction with the accompanying drawings.

The application provides a tumor sample specific inhibitor resistant to platinum drugs and combination drugs containing the platinum drugs and application thereof in treating colorectal cancer related drug resistant tumors.

The inventors first constructed a number of colorectal cancer cells resistant to oxaliplatin with different genetic backgrounds. By comparing with normal non-drug resistant cancer cells, it was found that the knockout of PLK4 gene in drug resistant cells can more significantly kill drug resistant cells, i.e., drug resistant cells are more sensitive to the knockout of PLK 4. PLK4 gene knockout blocks the progression of cell division to a greater extent in drug resistant cells.

Based on this, the inventors used a PLK4 specific inhibitor, compound CFI-400945, or inactivated PLK4 gene function by genetic means such as CRISPR, as a drug candidate for the treatment of colorectal cancer-related drug resistance. We found that compound CFI-400945 or CRISPR knockdown PLK4, which exhibited more pronounced tumor cell killing activity in a variety of colorectal cancer cells resistant to oxaliplatin than non-resistant cells; CFI-400945 treatment resulted in more pronounced cell cycle arrest of drug resistant cells; CFI-400945 also showed more remarkable inhibition effect on oxaliplatin-resistant colorectal cancer tumors in a mouse in vitro xenograft model. Therefore, the application proposes to use the PLK4 inhibitor CFI-400945 as a therapeutic drug for colorectal cancer resistant to oxaliplatin and a combination drug containing oxaliplatin clinically, and provide a new effective treatment and medication strategy for specific drug-resistant patient groups under corresponding conditions.

The inventor proposes to use PLK4 targeted drug CFI-400945 as a therapeutic drug for colorectal cancer resistant to oxaliplatin and a combination drug containing oxaliplatin clinically, and provide a new effective treatment and medication strategy for specific drug-resistant patient groups under corresponding conditions.

Thus, the present application provides the use of a PLK4 targeted drug in the manufacture of a medicament for the treatment of platinum drug resistant cancers.

IN some embodiments, the PLK4 targeting drug is CFI-400945, centrinone (LCR-263) (CAS No.: 1798871-30-3), centrinone-B (LCR-323) (CASNo.: 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, cancer Chemother Pharmacol, 2020) (CAS: N/A), (3-arylvinyl-1H-indazol-6-yl) pyrimidine-2-amine (Zhio Liu, et al, advances, 2017) (CAS: N/A), (3H-indazol-6-yl) pyrimidine-2-amine (Zhio Liu, et al, CAS: N/A), (3-H-indazol-6-yl) (RAdo et al, 2012) (CAS: N/A), YLZ-F5 (Yongxia zha, et al).

In some embodiments, the PLK4 targeting agent is a CRISPR reagent for inactivating PLK4 gene function.

In some preferred embodiments, the PLK4 inhibitor is CFI-400945, or pharmaceutically acceptable salts and crystalline forms thereof.

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 crystalline forms of CFI-400945 disclosed in CN201480064037.9 can be used.

In some preferred embodiments, the platinum-based drug is oxaliplatin. The platinum-based drugs also include cisplatin, carboplatin, nedaplatin, cisplatin, cycloplatin, lobaplatin, satraplatin, and platinum oxalate.

In some preferred embodiments, the platinum drug resistant cancer is colorectal cancer.

The platinum-based drug resistant cancer that can be treated with the PLK4 inhibitor can also be lung cancer, breast cancer, ovarian cancer, osteosarcoma, neuroblastoma, cervical cancer, endometrial cancer, vulval cancer, vaginal cancer, fibrosarcoma, prostate cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, esophageal cancer, chronic granulocytic leukemia, gastrointestinal sarcoma, glioblastoma multiforme, fibrohistiocytoma, round cell tumor sarcoma, synovial sarcoma, oropharyngeal cancer, lymphoma, sarcoma, brain cancer, paratumor, anogenital cancer, germ cell tumor, glioma, or malignant mesothelioma, etc.

In some embodiments, the PLK4 inhibitor, e.g., CFI-400945, may be administered to a subject up to about 0.05mg, 0.1mg, 0.2mg, 0.5mg, 1mg, 2mg, 5mg, 10mg, 50mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once a day, twice a day, three times a day, four times a day, once a week, twice a week, or three times a week (or no more than three times a week).

In some embodiments, the minimum dose level of PLK4 inhibitor for achieving 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 20000ng/kg subject body weight.

In some embodiments, the maximum dosage level of PLK4 inhibitor for achieving treatment may not exceed 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 20000ng/kg subject body weight.

In some embodiments, CFI-400945 is administered at a dosage of 10-20000ng/kg subject body weight/day, preferably CFI-400945 is administered at a dosage of about 7.5-10mg/kg subject body weight/day, more preferably CFI-400945 is administered at a dosage of about 10mg/kg subject body weight/day.

In some embodiments, the PLK4 inhibitors for effecting treatment may be administered to a subject by a variety of routes including, but not limited to, oral, topical, buccal, sublingual, pulmonary, transdermal, transmucosal, and subcutaneous, intraperitoneal, intravenous, and intramuscular injection or administration through the digestive tract in liquid or solid dosage forms.

In some embodiments, the PLK4 inhibitors used to effect treatment may be formulated into pharmaceutical compositions in solid dosage forms. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage forms may be, for example, fast-melt dosage forms, controlled release dosage forms, freeze-dried dosage forms, delayed release dosage forms, extended release dosage forms, pulsed release dosage forms, mixed immediate release and controlled release dosage forms, or combinations thereof.

In some embodiments, the PLK4 inhibitor may be formulated into a pharmaceutical composition comprising a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

The pharmaceutical compositions 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 crosslinked polyvinylpyrrolidone, microcrystalline celluloses, such as Avicel PH101 and Avicel PH102, microcrystalline celluloses and silicified microcrystalline celluloses (ProSolv SMCC) ^TM ). Suitable lubricants, including agents that act on the flowability of the powder to be compacted, may include colloidal silica, such as Aerosil 200, talc, stearic acid, magnesium stearate, calcium stearate and colloidal silica. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame potassium. Examples of flavoring agents are Magnasweet (trademark of MAFCO), bubble gum flavor, fruit flavor, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and salts thereof, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethanol or benzyl alcohol, phenolic compounds such as phenol or quaternary compounds such as benzalkonium chloride.

Suitable diluents may include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose such as Avicel PH101 and Avicel PH102; lactose such as lactose monohydrate, lactose anhydrous and Pharmatose DCL21; dibasic calcium phosphate such as Emcommess; mannitol; starch; sorbitol; sucrose and glucose.

Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, corn starch and modified starches, crosslinked sodium carboxymethylcellulose, crosslinked povidone, sodium starch glycolate, and mixtures thereof.

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, and 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 couple may be present.

In some embodiments, the PLK4 inhibitors for effecting treatment may be formulated into pharmaceutical compositions for delivery by any suitable route. For example, the pharmaceutical composition may be administered orally, intravenously, intramuscularly, subcutaneously, topically. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders and granules.

In some embodiments, the PLK4 inhibitors for effecting treatment may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating, and compressing or dissolving ingredients appropriate for the desired formulation.

Pharmaceutical compositions comprising PLK4 inhibitors may be adapted for administration by any suitable route, for example by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal and intrathecal) route. Such pharmaceutical compositions may be prepared by any method known in the pharmaceutical arts, for example by combining the active ingredient with a carrier or excipient.

Pharmaceutical compositions suitable for oral administration may be presented in discrete unit form, such as capsules or tablets; powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

Tablets and capsules for oral administration may be in unit dosage form and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, such as potato starch; or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated according to methods well known in conventional pharmaceutical practice. Oral liquid preparations 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 vehicle as a dry product before use. Such liquid formulations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; a non-aqueous carrier (possibly including edible oils), such as almond oil, oily esters such as glycerol, propylene glycol or ethanol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.

Specific materials and sources thereof used in embodiments of the present application are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present application.

Cell biology techniques used in practice include cell culture, cell transfection, cell infection, cell counting, etc., and mouse techniques include subcutaneous tumor and mouse lavage, which are usually performed according to conventional methods unless otherwise indicated.

Abbreviations:

HCT116-Mock is HCT116 cells without any treatment;

HCT116-Vector HCT116 cells infected with HCT116 with the lentiCRISPR-v2 Vector;

HCT116-AAVS1 is HCT116 cells from which HCT116 knocks out AAVS1 via CRISPR/Cas9 system, AAVS1 is currently defined as a safe harbor site, i.e., no significant functional impact on genome after knockout;

HCT116-ΔTP53 ^OR -1 and HCT 116-. DELTA.TP 53 ^OR -2 is a drug-resistant cell strain constructed by knocking out TP53 from HCT116 through CRISPR/Cas9 system, delta represents knocking out, OR is oxaliplatin resistance, and the gene isTwo pairs of sgRNAs are respectively designed, namely named-1 and-2;

HCT116-ΔSLC43A2 ^OR -1 and HCT116- ΔSLC43A2 ^OR -2 is a drug-resistant cell strain constructed by HCT116 knocking out SLC43A2 through CRISPR/Cas9 system, "delta" represents knocking out, OR is oxaliplatin resistance, and two pairs of sgRNAs are respectively designed due to genes, namely named-1 and-2;

HCT116-ΔCDKN1A ^OR -1 and HCT116- ΔCDKN1A ^OR 2 is a drug-resistant cell strain constructed by knocking out CDKN1A of HCT116 through a CRISPR/Cas9 system, delta represents knocking out, OR is oxaliplatin resistance, and two pairs of sgRNAs are respectively designed due to genes, namely named-1 and-2;

HCT116 ^OR 、DLD1 ^OR 、HCT8 ^OR and HT29 ^OR The strain is an obtained oxaliplatin resistant strain constructed by gradually increasing the concentration of oxaliplatin drug in colon cancer wild type cells.

First, a plurality of colon cancer cells resistant to oxaliplatin with different genetic backgrounds were constructed. Drug-resistant cell strain HCT 116-delta TP53 constructed by knocked-out genes respectively ^OR 、HCT116-ΔSLC43A2 ^OR And HCT116- ΔCDKN1A ^OR (shown in FIG. 1) and the acquired drug-resistant strain HCT116 constructed by gradually increasing the concentration of oxaliplatin drug by normal colon cells respectively ^OR 、DLD1 ^OR 、HCT8 ^OR And HT29 ^OR (shown in FIG. 2).

By comparison 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 could kill drug resistant cells more significantly, i.e., drug resistant cells were more susceptible to the knocking out of PLK4 (shown in FIGS. 3 and 4).

PLK4 protein localizes to the centrosome and regulates centrosome replication during the cell cycle. We found that PLK4 gene knockout blocked the progression of cell division to a greater extent in drug-resistant cells relative to normal non-drug-resistant cancer cells (shown in FIG. 5). Cells knocked out of PLK4 formed short bipolar spindles, and oxaliplatin-resistant strain cells exhibited spindle collapse, arrest in mitosis or slipped out of mitosis without undergoing later stages, failed chromosome segregation, and eventually resulted in growth arrest, as compared to non-drug-resistant strain Vector group (shown in fig. 6).

Based on the experimental results, a PLK4 specific inhibitor, namely a compound CFI-400945, is used as a drug candidate for treating colorectal cancer related drug resistance. We found that compound CFI-400945 exhibited more pronounced tumor cell killing activity in a variety of colorectal cancer cells resistant to oxaliplatin than non-resistant cells (shown in figures 7 and 9). In terms of cell morphology, it is evident that oxaliplatin-resistant cell lines were sensitive to CFI-400945, and that the oxaliplatin-resistant cell lines exhibited significant cell morphology deterioration and membrane fusion compared to non-resistant cells (shown in fig. 8). CFI-400945 treatment resulted in more pronounced cell cycle arrest of drug resistant cells (shown in figure 10). Cells treated with CFI-400945 formed short bipolar spindles, and oxaliplatin-resistant strain cells exhibited spindle collapse, arrest in mitosis or slipped out of mitosis without undergoing later stages, resulting in failure of chromosome segregation, and eventually in growth arrest, compared to non-resistant strain Vector groups. (shown in FIG. 11).

CFI-400945 also showed more significant inhibition of oxaliplatin-resistant colorectal tumors in a mouse in vivo xenograft model. The mice experiments were divided into six groups, subcutaneous tumor non-drug resistant cells and oxaliplatin resistant cells. 6 days after the mice were subcutaneously inoculated with tumors, HCT116-Vector, HCT116- ΔSLC43A2 were treated with vehicle and CFI-400945, respectively ^OR And HCT116 ^OR The mice in the experimental group were subjected to gastric lavage treatment with CFI-400945 at a drug concentration of 7.5 mg/kg/day. At the end of the experiment, HCT 116-. DELTA.SLC43A 2 compared to HCT116-Vector-CFI-400945 ^OR -CFI-400945 and HCT116 ^OR The tumor volume and weight in CFI-400945 were significantly reduced (shown in FIG. 12).

Based on the experiment of FIG. 12, we readjust the dose of CFI-400945 with a CFI-400945 drug concentration of 10 mg/kg/day. At this dose, HCT116 was compared to HCT116-Vector-CFI-400945 at the end of the experiment ^OR- There was a more significant decrease in tumor volume and weight in CFI-400945 (shown in fig. 13).

In the following examples:

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 (American Type Culture Collection, ATCC) and were cultured in DMEM medium (HCT 116 and HT29 cells) or RPMI 1640 medium (DLD 1 and HCT8 cells) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃and 5% CO ₂ Culturing.

DMEM is purchased from BI, cat#06-1055-57-1ACS. RPMI-1640 is purchased from BI, cat #01-100-1ACS.

Oxaliplatin (oxaliplatin) was purchased from the national food and drug verification institute (Cat# 100584), CFI-400945 (CAS# 1616420-30-4) was purchased from MCE (Cat#HY-12300B), nude mice used in the examples were purchased from Fukang Biotechnology Co., ltd. Beijing, and sgRNA sequences used in the examples were synthesized by Song Biotechnology Co., ltd. In Table 1.

TABLE 1 sgRNA target sequences

Example 1: oxaliplatin drug-resistant cell strain constructed by knocking out drug-resistant related genes

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 the gene using the sgrnas. The HCT116 knockout gene cell lines show obvious drug-resistant cell populations after 6 days of treatment with 5 mu M oxaliplatin. That is, mutations or functional inactivation of TP53, SLC43A2, and CDKN1A may result in a chemotherapeutic drug resistant phenotype. For calculating IC ₅₀ Is based on MTT colorimetry. Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit in cells, whereas dead cells do not. Formation ofThe number of MTT crystals of (2) is proportional to the number of cells. The number of living cells was then measured based on the absorbance values. Cells (4X 10 per well) ³ And 3) were plated in 96-well plates, cells were treated with oxaliplatin in DMSO for 3 days after attachment, 3 duplicate wells were set for each drug concentration and the same volume of DMSO was added as a blank. Then, 10. Mu.L of MTT (5 mg/mL) was added to the well wall above the 96-well plate, gently shaken well, and placed in a cell incubator for continuous culture for 4 hours. Thereafter, the medium was gently aspirated, 100. Mu.L of DMSO was added to each well, and the mixture was gently shaken for 5min to promote the crystallization to be sufficiently dissolved. Determination of cell viability by absorbance at 490nm using a BioTek spectrophotometer (Gene Company Limited), calculation of IC using Prism9 software ₅₀ Values. The upper graph of FIG. 1 shows the cell growth curves of the knockout gene resistant cell lines of HCT116 measured by MTT method under the treatment of oxaliplatin with different concentrations, and the lower graph shows the IC of oxaliplatin on the knockout gene resistant cell lines of HCT116 ₅₀ A histogram of values.

Example 2: acquired oxaliplatin resistant cell lines of colon cancer cells with different genetic backgrounds

Colon cancer cells with four different genetic backgrounds of HCT116, DLD1, HCT8 and HT29 are treated by gradually increasing the concentration of oxaliplatin medicament (gradually increasing the concentration of oxaliplatin from 1 mu M of oxaliplatin), and the acquired drug-resistant strain HCT116 is constructed after about 2-3 months ^OR 、DLD1 ^OR 、HCT8 ^OR And HT29 ^OR . The final oxaliplatin concentrations of HCT116, DLD1, HCT8 and HT29 were 45. Mu.M, 80. Mu.M, 50. Mu.M and 35. Mu.M, respectively, and after about one week of treatment with each final concentration, the normal HCT116, DLD1, HCT8 and HT29 cells of each of the corresponding control groups showed significant cell death, and it was evident that all four cell lines were significantly successful in constructing the acquired drug resistant strain (FIG. 2). ", is p" compared with the non-drug-resistant strain control group<0.01。

Example 3: HCT116 each oxaliplatin resistant cell line is susceptible to inactivation by PLK4

10 cell lines HCT116-Mock, HCT116-Vector, HCT116-AAVS1, HCT116- ΔTP53 ^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 control group (sgCtrl: empty Vector without sgRNA) and the experimental group (sgPLK 4-1, sgPLK4-2: CRISPR knock-out two sgRNAs of PLK4; sgPLK1-1, sgPLK1-2: CRISPR knock-out two sgRNAs of PLK 1) were divided.

HCT116 cells were lysed in RIPA buffer (beyotidme) containing phosphatase inhibitor (meiumbio #mb 12707) and protease inhibitor (meiumbio #mb 26780) in an environment of 4 ℃ for 15 min. After that, the mixture was centrifuged at 14,000rpm at 4℃for 10 minutes and the supernatant was taken. The protein supernatant was mixed with loading buffer and then the protein was separated on a 10% bis-tris polyacrylamide gel. The proteins in the gel were transferred to Nitrocellulose (NC) membrane (Pall Corporation # 27574625) and blocked with 5% skim milk (DifcoTM Skim Milk # 4296916). PLK4 and PLK1 protein expression was detected using a chemiluminescent imaging system (Tanon-5200) after incubation with primary antibodies (PLK 4; cell Signaling Technology; cat#71033 and PLK1; proteintech; cat#10305-1-AP) and secondary antibodies (Goat anti-Rabbit IgG; thermo Fisher Scientific; cat# 31460), respectively. Protein expression levels of PLK4 or PLK1 could be successfully reduced in HCT116 cells after infection with the corresponding CRISPR sgRNA virus (fig. 3).

Cell counts were performed with a blood cell counting plate 11 days after the above 10 cell lines were infected with the corresponding sgRNA viruses. When PLK4 was knocked out, the number of surviving cells was significantly less in all the tested drug resistant cells than in non-drug resistant cells, indicating that drug resistant cell lines were more susceptible to PLK4 functional inactivation (FIG. 3); while knockout of PLK1 only has a weak killing effect in individual drug-resistant cells (fig. 3, below), indicating that PLK4 is more suitable as a molecular target against drug resistance than PLK 1. ". Times." is p <0.01 compared to Mock, vector and AAVS1 within each group.

Example 4: DLD1, HCT8 and HT29 oxaliplatin resistant cell lines are susceptible to inactivation by PLK4

DLD1, HCT8 and HT29 oxaliplatin resistant and non-resistant cell lines were divided into control (sgCtrl: empty Vector without sgRNA) and experimental groups (sgPLK 4-1, sgPLK4-2: CRISPR knockdown of two sgRNAs of PLK 4), and cell growth was observed after infection with the corresponding sgRNA viruses, and cell counts were performed using a hemocytometer. After knockout of PLK4, the number of surviving resistant cells is significantly less than non-resistant cells. That is, DLD1, HCT8 and HT29 resistant cell lines were also more susceptible to PLK4 functional inactivation as concluded in example 3 (fig. 4). ". Times." is p <0.05 compared to the non-drug-resistant strain control group; "×" is p <0.01 compared to the non-drug resistant strain control group.

Example 5: PLK4 gene knockout blocks the progression of cell division to a greater extent in drug-resistant cells

In this example, four cell lines of HCT116 (Vector, ΔSLC43A2 ^OR 、ΔTP53 ^OR 、HCT116 ^OR ) The control group (sgAAVS 1) and the experimental group (sgPLK 4-1) were divided. 12 days after infection, each treatment group was treated with 75. Mu.M 5-bromodeoxyuridine (BrdU) for 1h. Cells were thoroughly digested and washed with PBS. 90% ethanol was added dropwise under vortexing to fix cells. Light was protected overnight at 4 ℃. The samples were resuspended and washed with PBS. 0.5mL of 2M HCl (0.5% Triton X-100) was added, vortexed and incubated for 30 minutes at room temperature, followed by washing with PBS. 1mL of 100mM sodium borate solution (pH=8.5) was added, the cells resuspended and centrifuged at 2000rpm for 5 minutes. The supernatant was discarded, 1ml of 3% BSA blocking solution (PBS-0.5% Tween20, PBST) was added, incubated for 30 minutes at room temperature, and then the supernatant was removed by centrifugation. mu.L of BrdU primary antibody (Cell Signaling Technology #5292; 1:200) was added and incubated for one hour at room temperature. PBST wash, 100. Mu.L of mouse fluorescent secondary antibody (Thermo Fisher Scientific #A-11001; 1:500) was added. Light was protected and incubated at room temperature for 30 minutes, then washed with PBST. Add 350. Mu.L PI/RNase Staining Buffer (BD # 550825) and incubate at room temperature in the dark for 30 minutes. Samples were filtered using a 200 mesh nylon membrane, run on a Flow cytometer (BD LSRFortessa), and analyzed for cell cycle distribution using Flow jo_v10 software. After knockout of PLK4, each drug-resistant cell cycle was significantly arrested in G2/M phase, delaying the mitotic progression of the cells compared to the non-drug-resistant cell group (fig. 5).

Example 6: PLK4 gene knockout leads to spindle collapse in drug resistant cells

In this example, four cell lines of HCT116 (Vector, ΔSLC43A2 ^OR 、ΔTP53 ^OR 、HCT116 ^OR ) The control group (sgAAVS 1) and the experimental group (sgPLK 4-1) were divided. 10 days after infection, cells were seeded in 24-well plates with circular slides. After cell attachment, cells were treated with 2mM thymidine for 16 hours, and then the normal medium was changed for further culture for 8 hours. This procedure was repeated once, and cells were treated with 100ng/mL Nocodazole (Nocodazole) for 5 hours to complete the cell synchronization treatment. Thereafter, cells were fixed at five time points: 0 minutes, 30 minutes, 45 minutes, 60 minutes and 90 minutes. Cells were washed 3 times for 3 minutes with PBS prior to fixation. The slides were then fixed with 3.7% paraformaldehyde for 10 minutes, washed 3 times with PBS for 3 minutes each. Cell membranes were permeabilized with 0.1% Triton X-100 (in PBS) for 2 min at room temperature and washed 3 times with PBS. Slides were blocked with Bovine Serum Albumin (BSA) for 30 minutes at room temperature. Diluted primary antibodies (α -tubulin 1:100, protein # 66031-1-Ig) were added to each slide, the slides were placed in a wet box, and incubated overnight at 4 ℃. Slides were immersed in PBST for 3 washes, 3 minutes each, followed by addition of fluorescent secondary antibodies (goat anti-mouse IgG,1:500,Thermo Fisher Scientific#A-11001) and incubation in wet cartridges for 1 hour at room temperature. Slides were immersed in PBST for 3 washes for 3 minutes each. DAPI was added for 5min incubation and then washed 3 times for 5min each with PBST. The slide was blotted with a piece of blotter paper and sealed with an anti-fluorescence quencher. Images were collected under a confocal microscope (Leica TCS SP 8). After knockout of PLK4, each resistant cell exhibited spindle collapse, folding, and slipping compared to the non-resistant cell group (fig. 6), resulting in chromosome segregation failure, ultimately leading to growth arrest.

Example 7: HCT116 oxaliplatin resistant cell lines are sensitive to CFI-400945

The cell lines used in this example were the same as in example 3 and were counted using a blood cell counting plate, and it was concluded that the drug resistant cell lines were more sensitive to the PLK4 inhibitor CFI-400945 as in example 3. In other words, CFI-400945 was able to kill oxaliplatin resistant cell lines more effectively (fig. 7). This example uses CFI-400945 at a concentration of 12nM. ". Times." is p <0.01 compared to Mock, vector and AAVS1 within each group.

Example 8: cell morphology shows that each oxaliplatin-resistant cell line of HCT116 is sensitive to CFI-400945

Ten cell lines in this example were plated at the same cell amount as in example 3, and after cell adhesion, cells were treated with CFI-400945 of 12nM for 7 days, the PLK4 inhibitor CFI-400945 had a higher killing power against the drug-resistant cell line than the non-drug-resistant cell line, and the cells were inferior in morphology and membrane fusion (fig. 8).

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

DLD1, HCT8 and HT29 oxaliplatin resistant and non-resistant cell lines were treated with CFI-400945 at concentrations of 10nM, 15nM and 15nM, respectively, and cell counts were performed using a blood cell counting plate to draw a conclusion similar to that of example 7 that resistant cell lines were more sensitive to the PLK4 inhibitor CFI-400945, or that CFI-400945 was more effective in killing oxaliplatin resistant cell lines (FIG. 9). ". Times." is p <0.05 compared to the non-drug-resistant strain control group; "×" is p <0.01 compared to the non-drug resistant strain control group.

Example 10: CFI-400945 treatment, oxaliplatin-resistant cells developed a more pronounced cell cycle arrest

Four cell lines in this example, HCT116 ^OR 、HCT116-ΔTP53 ^OR 、HCT116-ΔSLC43A2 ^OR And HCT116-Vector were treated with DMSO and 12nM CFI-400945, respectively, for 7 days, for details of experimental procedures as described in example 5.CFI-400945 treatment significantly retarded the mitotic progression of the cells by blocking the individual drug-resistant cell cycle in the G2/M phase compared to the non-drug-resistant cell group (fig. 10).

Example 11: treatment with CFI-400945 results in spindle collapse in drug resistant cells

Four cell lines in this example, HCT116 ^OR 、HCT116-ΔTP53 ^OR 、HCT116-ΔSLC43A2 ^OR And HCT116-Vector were treated with DMSO and 12nM CFI-400945, respectively, for 7 days, for details of the experimental procedure described in example 6. After CFI-400945 treatment, compared with a non-drug-resistant cell group, each drug-resistant cell has the phenomena of spindle body collapse, folding, sliding and the like,causing chromosome segregation failure and ultimately resulting in growth arrest (fig. 11).

Example 12: in the xenograft model of mice, oxaliplatin resistant tumors developed more remarkable volume and weight reductions after treatment with CFI-400945

The mice experiments were divided into six experimental groups, namely non-oxaliplatin resistant cell lines: HCT116-Vector, oxaliplatin resistant cell line: HCT 116-DeltaSLC 43A2 ^OR And HCT116 ^OR Respectively at 3 x10 ⁶ Subcutaneous tumor of mice was inoculated on both the left and right sides of the skin with a tumor volume of about 100mm ³ At time (day 6 post-tumor), HCT116-Vector, HCT116- ΔSLC43A2 was treated with solvent control (DMSO) and CFI-400945, respectively ^OR And HCT116 ^OR Mice in the experimental group were subjected to intragastric treatment with CFI-400945 at a drug concentration of 7.5 mg/kg/day for 5 days per week. At the end of the experiment (30 days after tumor seeding), the tumor was peeled off, the tumor volume and weight were weighed, and the tumor volume was measured with a vernier caliper and calculated using the following formula: volume= (length width)/2. The CFI-400945 treatment resulted in a more significant reduction in tumor volume and weight in each resistant cell group compared to the non-resistant cell group (fig. 12). Wherein the HCT116-Vector group, HCT 116-delta SLC43A2 ^OR And HCT116 ^OR The tumor volume inhibition rates of the groups 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 tumor suppression rate = (control mean volume-experimental mean volume)/control mean volume x 100%. Tumor mass inhibition rate= (control group average mass-experimental group average mass)/control group average mass x 100%. n=17 (Vector-Vector), n=18 (Vector-CFI-400945), n=15 (Δslc43 A2) ^OR -1-Vehicle)，n＝15(ΔSLC43A2 ^OR -1-CFI-40094)，n＝17(HCT116 ^OR -Vehicle)，n＝17(HCT116 ^OR -CFI-400945). ", is p compared with the control group<0.05; ", p" is compared with the control group<0.01。

Example 13: in the mouse xenograft model, tumors formed by acquired oxaliplatin-resistant cells were more significantly reduced in volume and weight by treatment with high concentrations of CFI-400945.

A mouseThe experiments were divided into four experimental groups, namely non-oxaliplatin resistant cell lines: HCT116-Vector, oxaliplatin resistant cell line: HCT116 ^OR Respectively at 3 x10 ⁶ Subcutaneous tumor of mice was inoculated on both the left and right sides of the skin with a tumor volume of about 100mm ³ At time (day 6 post-tumor), HCT116-Vector and HCT116 were treated with solvent control (DMSO) and CFI-400945, respectively ^OR Mice in the experimental group were subjected to intragastric treatment with CFI-400945 at a drug concentration of 10 mg/kg/day for 5 days per week. At the end of the experiment (22 days after tumor seeding), the tumor was peeled off, the tumor volume and weight were weighed, and the tumor volume was measured with a vernier caliper and calculated using the following formula: volume= (length width)/2. CFI-400945 treatment resulted in a more significant reduction in tumor volume and weight in the resistant cell group compared to the non-resistant cell group (FIG. 13) where HCT116-Vector groups and HCT116 ^OR The tumor volume inhibition rates of the groups were 25.6% and 83.7%, respectively, and the tumor weight inhibition rates were 57.3% and 79%, respectively. Compared to HCT116 in example 12 (FIG. 12) ^OR The inhibition rate of the tumor volume of the group is 42 percent, the inhibition rate of the tumor weight is 36.4 percent, and the effect of the CFI-400945 medicine concentration of the group at the dosage of 10 mg/kg/day is more obvious. Tumor volume tumor suppression rate = (control mean volume-experimental mean volume)/control mean volume x 100%. Tumor mass inhibition rate= (control group average mass-experimental group average mass)/control group average mass x 100%. n=4 (Vector-Vector), n=5 (Vector-CFI-400945), n=5 (HCT 116) ^OR -Vehicle)，n＝5(HCT116 ^OR -CFI-400945). ", is p compared with the control group<0.05; ", p" is compared with the control group<0.01; ", p" is compared to the control group<0.0001。

The present application has been described in terms of several embodiments, but the description is illustrative and not restrictive, and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the described embodiments. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are possible. Any feature of any embodiment may be used in combination with, or in place of, any other feature of any other embodiment, unless expressly limited otherwise.

Claims

Use of a plk4 targeted drug in the manufacture of a medicament for the treatment of platinum drug resistant cancer.
2. The use of claim 1, wherein the platinum-based drug resistant cancer is colorectal cancer, lung cancer, breast cancer, ovarian cancer, osteosarcoma, neuroblastoma, cervical cancer, endometrial cancer, vulval cancer, vaginal cancer, fibrosarcoma, prostate cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, esophageal cancer, chronic granulocytic leukemia, gastrointestinal sarcoma, glioblastoma multiforme, fibrohistiocytoma, round cell sarcoma, synovial sarcoma, oropharyngeal cancer, lymphoma, sarcoma, brain cancer, accessory tumors, anogenital cancer, germ cell tumor, glioma, or malignant mesothelioma.
3. The use of claim 2, wherein the platinum-based drug resistant cancer is colorectal cancer.
4. The use according to claim 1, wherein the platinum-based drug is selected from oxaliplatin, cisplatin, carboplatin, nedaplatin, eplatin, leplatin, cycloplatin, lobaplatin, satraplatin and platinum oxalate.
5. The use according to claim 4, wherein the platinum-group drug is oxaliplatin.
6. The use of any one of claims 1 to 5, wherein the PLK4 targeting drug is a PLK4 inhibitor or a gene editing drug.
7. The use of claim 6, wherein 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, yl t-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 crystalline forms thereof.
8. The use of claim 7, wherein the PLK4 inhibitor is CFI-400945, or pharmaceutically acceptable salts and crystalline forms thereof.
9. The use of claim 8, wherein CFI-400945 is administered at a dose of 10-20000ng/kg subject body weight/day, preferably CFI-400945 is administered at a dose of about 7.5-10mg/kg subject body weight/day, more preferably CFI-400945 is administered at a dose of about 10mg/kg subject body weight/day.
10. The use of claim 6, wherein the gene editing drug is a CRISPR reagent that inactivates PLK4 gene function.