CN114908158B

CN114908158B - Use of CDK1 in diagnosis and treatment of advanced gastrointestinal stromal tumors

Info

Publication number: CN114908158B
Application number: CN202110172517.9A
Authority: CN
Inventors: 王跃祥; 芦晓晶
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2024-04-30
Anticipated expiration: 2041-02-08
Also published as: CN114908158A

Abstract

The present invention relates to the use of CDK1 in the diagnosis and treatment of advanced gastrointestinal stromal tumors. Specifically, the invention provides the use of a CDK1 gene, mRNA, cDNA, or protein or detection reagent thereof as (i) a marker for the detection of advanced gastrointestinal stromal tumors; and/or (ii) for the preparation of a diagnostic reagent or kit for the detection of advanced gastrointestinal stromal tumors, and the inhibitors of the CDK1 gene or protein thereof of the invention may optionally be used in combination with tyrosine kinase inhibitors, and/or optionally other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumors, and have a significant synergistic effect on the treatment of advanced gastrointestinal stromal tumors.

Description

Use of CDK1 in diagnosis and treatment of advanced gastrointestinal stromal tumors

Technical Field

The present invention relates to the field of oncology and diagnostics. More particularly, the invention relates to the use of CDK1 in the diagnosis and treatment of advanced gastrointestinal stromal tumors.

Background

Gastrointestinal stromal tumors are the most common mesenchymal-derived tumors of the gastrointestinal tract. Gastrointestinal stromal tumors are classified into primary and metastatic gastrointestinal stromal tumors according to the presence or absence of metastasis, primary gastrointestinal stromal tumors are classified into low-risk, medium-risk and high-risk gastrointestinal stromal tumors according to pathological indexes (tumor size, mitosis per high-power visual field, anatomical position, etc.), and if high-risk stromal tumors refer to the high-risk of occurrence of metastasis and recurrence. High-risk gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors are collectively referred to as advanced gastrointestinal stromal tumors. Clinically, the treatment principles and schemes of the interstitial tumors with different risk levels are different. After receiving standard treatment protocols, the clinic still has partial moderate-risk and even low-risk gastrointestinal stromal tumors with poor curative effects (manifested as tumor recurrence and metastasis). There is therefore a great need in the art to develop targets for gastrointestinal stromal tumors that have the value to assess patient risk levels.

In addition, advanced gastrointestinal stromal tumors are often misdiagnosed as other digestive tract tumors (e.g., digestive tract smooth muscle tumors, digestive tract schwannomas, etc.), so there is a need in the art to develop targets with differential diagnosis of advanced gastrointestinal stromal tumors.

And, currently, most patients develop drug resistance for gastrointestinal stromal tumor patients receiving targeted therapy.

Thus, there is an urgent need in the art to develop new targets with diagnostic and therapeutic effects and new methods to overcome drug resistance and sensitivity.

Disclosure of Invention

The object of the present invention is to provide new targets with diagnostic and therapeutic effects and new methods to overcome drug resistance and sensitivity.

In a first aspect the invention provides the use of a CDK1 gene, mRNA, cDNA, or protein, or a detection reagent thereof, (i) as a marker for the detection of advanced gastrointestinal stromal tumors; and/or (ii) for the preparation of a diagnostic reagent or kit for the detection of advanced gastrointestinal stromal tumors.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

In another preferred embodiment, the CDK1 gene, mRNA, cDNA or protein is derived from a mammal, preferably a rodent (e.g.mouse, rat), primate or human, more preferably a patient diagnosed with advanced gastrointestinal stromal tumor.

In another preferred embodiment, the CDK1 gene, mRNA, cDNA, or protein is derived from a patient with advanced gastrointestinal stromal tumor.

In another preferred embodiment, the CDK1 gene has accession number NG_029877.1.

In another preferred embodiment, the CDK1 mRNA has accession number NM-001786.

In another preferred embodiment, the CDK1 protein has accession number NP-001777.

In another preferred embodiment, the assay is a tissue sample assay.

In another preferred embodiment, the detection comprises immunohistochemistry, immunoblotting, co-immunoprecipitation and fluorescent quantitative PCR methods.

In another preferred embodiment, the detection is of early stage gastrointestinal stromal tumor tissue, or late stage gastrointestinal stromal tumor tissue samples.

In another preferred embodiment, the detection reagent comprises an antibody specific for CDK1, a specific binding molecule for CDK1, a specific amplification primer, a probe or a chip.

In another preferred embodiment, the detection reagent is selected from the group consisting of: antibodies, primers, probes, sequencing libraries, nucleic acid chips (e.g., DNA chips), protein chips, or combinations thereof.

In another preferred embodiment, the CDK1 protein or specific antibody or specific binding molecule thereof is conjugated or provided with a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the antibody specific for CDK1 is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the CDK1 protein further comprises a derivative of CDK1 protein.

In another preferred embodiment, the derivative of the CDK1 protein comprises a modified CDK1 protein, a protein molecule having an amino acid sequence homologous to a native CDK1 protein and having native CDK1 protein activity, a fusion protein comprising the amino acid sequence of a CDK1 protein.

In another preferred embodiment, the modified CDK1 protein is a pegylated CDK1 protein.

In another preferred embodiment, the "protein molecule having an amino acid sequence homologous to a native CDK1 protein and having native CDK1 protein activity" means that its amino acid sequence has more than or equal to 85% homology, preferably more than or equal to 90% homology, more preferably more than or equal to 95% homology, most preferably more than or equal to 98% homology to CDK1 protein; and a protein molecule having native CDK1 protein activity.

In another preferred embodiment, the diagnostic reagent or kit is also used to distinguish between early stage and late stage gastrointestinal stromal tumors.

In a second aspect, the invention provides a diagnostic kit for the detection of advanced gastrointestinal stromal tumors, said kit comprising a container containing detection reagents for the detection of CDK1 genes, mRNA, cDNA or proteins; and a label or instructions stating that the kit is for detecting advanced gastrointestinal stromal tumors.

In another preferred embodiment, the detection of advanced gastrointestinal stromal tumor refers to determining the size of the likelihood of developing advanced gastrointestinal stromal tumor.

In another preferred embodiment, the judgment includes a preliminary judgment (prediction).

In another preferred embodiment, the assay reagent for detecting a CDK1 gene, mRNA, cDNA, or protein comprises:

(a) Specific antibodies against CDK1 proteins; and/or

(B) Specific primers for specific amplification of mRNA or cDNA of CDK 1.

In another preferred embodiment, the assay is a tissue sample assay.

In another preferred embodiment, the assay for CDK1 gene, mRNA, cDNA, or protein or assay reagents therefor may be used as a control or reference.

In another preferred embodiment, the diagnostic kit is also used to distinguish between early stage gastrointestinal stromal tumors and late stage gastrointestinal stromal tumors.

In another preferred embodiment, the label or instructions note that the kit is for:

(a) Detecting advanced gastrointestinal stromal tumors;

(b) Early stage gastrointestinal stromal tumors and late stage gastrointestinal stromal tumors are distinguished.

In another preferred embodiment, the subject is a human or non-human mammal.

In a third aspect the invention provides a method of detecting advanced gastrointestinal stromal tumors, the method comprising:

a) Providing a test sample from a subject;

b) Detecting the expression level E1 of CDK1 protein in the test sample; and

C) Comparing the expression level of CDK1 protein determined in step b) with a reference value,

Wherein an expression level of CDK1 protein in the sample that is higher than a reference value indicates that the subject has advanced gastrointestinal stromal tumor.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the test sample is a cell or tissue of an advanced gastrointestinal stromal tumor.

In another preferred embodiment, the reference value is a cut-off value.

In another preferred embodiment, the reference value is the relative expression level of CDK1 in the sample.

In another preferred embodiment, the reference value is 5 (RNA level).

In another preferred embodiment, the expression level of CDK1 RNA in the sample is detected by RT-PCR or transcriptome sequencing, and the expression level of CDK1 protein in the sample is detected by immunoblotting or immunohistochemistry.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a fourth aspect, the invention provides a method of determining a treatment regimen comprising:

a) Providing a test sample from a subject;

b) Detecting the expression level of CDK1 protein in the test sample; and

C) A treatment regimen is determined based on the expression level of CDK1 protein in the sample.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, when the level of expression of the CDK1 protein in the sample is greater than a reference value, indicating that the subject has advanced gastrointestinal stromal tumor, the treatment regimen comprises CDK1 inhibitor therapy, a therapy in which a CDK1 inhibitor is combined with a tyrosine kinase inhibitor.

In another preferred embodiment, the CDK1 inhibitor therapy, a CDK1 inhibitor in combination with a tyrosine kinase inhibitor is selected from the group consisting of:

CDK1 inhibitor therapy: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof;

tyrosine kinase inhibitor therapy: a small molecule compound selected from the group consisting of: imatinib, sunitinib, regorafenib, avapritinib (BLU-285), ripretinib (DCC-2618), or a combination thereof.

In another preferred embodiment, when the subject has advanced gastrointestinal stromal tumor, the treatment regimen further comprises a CDK1 inhibitor therapy, a therapy in which a CDK1 inhibitor is combined with a tyrosine kinase inhibitor; and other medicaments for treating advanced gastrointestinal stromal tumor.

In another preferred embodiment, the other agent for treating advanced gastrointestinal stromal tumors is selected from the group consisting of: imatinib, sunitinib, regorafenib, avaprinib (Avapritinib, BLU-285), repatinib (Ripretinib, DCC-2618), or a combination thereof.

In a fifth aspect the present invention provides the use of an inhibitor of the CDK1 gene or protein thereof for the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increase the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is selected from the group consisting of: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof.

In another preferred embodiment, the inhibitor comprises an inhibitor that inhibits expression of a CDK1 gene or protein thereof.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is selected from the group consisting of: RO-3306,

NU6027、Flavopiridol、AT7519、GW5074、Dinaciclib、JNJ-7706621、 AZD5438、BMS-265246、R547、CDKI-73、Alsterpaullone、SU9516、 Or a combination thereof.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the composition comprises a therapeutically effective amount of an inhibitor of the CDK1 gene or protein thereof, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the agent is administered by a regimen selected from the group consisting of: oral, intravenous, intramuscular, subcutaneous, sublingual, rectal infusion, nasal spray, oral spray, topical or systemic transdermal administration to the skin.

In another preferred embodiment, the formulation is selected from the group consisting of: tablets, capsules, injections, granules and sprays.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising:

(a1) Inhibitors of the CDK1 gene or protein thereof;

(a2) Tyrosine kinase inhibitors; and

(B) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises:

(c) Other drugs for preventing and/or treating advanced gastrointestinal stromal tumors.

In another preferred embodiment, the tyrosine kinase inhibitor is selected from the group consisting of: imatinib, sunitinib, regorafenib, avapritinib (BLU-285), ripretinib (DCC-2618), or a combination thereof.

In another preferred embodiment, the weight ratio of component (a 1) to component (a 2) is from 100:1 to 0.01:1, preferably from 10:1 to 0.1:1, more preferably from 2:1 to 0.5:1.

In another preferred embodiment, the content of the component (a 1) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of component (a 2) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of component (c) in the pharmaceutical composition is 1% -99%, preferably 10% -90%, more preferably 30% -70%.

In another preferred embodiment, the component (a 1) and the optional component (a 2) and the optional component (c) comprise 0.01 to 99.99wt%, preferably 0.1 to 90wt%, more preferably 1 to 80wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injectable dosage form, and an oral dosage form.

In another preferred embodiment, the oral dosage form comprises a tablet, a capsule, a film, and a granule.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained release dosage form, and a non-sustained release dosage form.

A seventh aspect of the invention provides a product combination comprising:

(i) A first pharmaceutical composition comprising (a) a first active ingredient which is an inhibitor of the CDK1 gene or protein thereof, and a pharmaceutically acceptable carrier; and

(Ii) A second pharmaceutical composition comprising (b) a second active ingredient which is a tyrosine kinase inhibitor, and a pharmaceutically acceptable carrier;

wherein the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions or the same pharmaceutical composition.

In another preferred embodiment, the weight ratio of component (i) to component (ii) is from 100:1 to 0.01:1, preferably from 10:1 to 0.1:1, more preferably from 2:1 to 0.5:1.

In another preferred embodiment, the content of component (i) in the product combination is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of component (ii) in the product combination is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the components (i) and (ii) comprise from 0.01 to 99.99wt%, preferably from 0.1 to 90wt%, more preferably from 1 to 80wt% of the total weight of the product combination.

In another preferred embodiment, the product combination further comprises a detection reagent for CDK1 or a kit thereof.

In another preferred embodiment, the kit comprises a container containing a detection reagent for detecting a CDK1 gene, mRNA, cDNA, or protein; and a label or instructions stating that the kit is for detecting advanced gastrointestinal stromal tumors.

An eighth aspect of the present invention provides a kit comprising:

(a1) A first container, and an inhibitor of a CDK1 gene or protein thereof, or a medicament containing an inhibitor of a CDK1 gene or protein thereof, located in the first container;

(b1) A second container, and a tyrosine kinase inhibitor, or a medicament containing a tyrosine kinase inhibitor, located in the second container.

In another preferred embodiment, the kit further comprises:

(c1) A third container, and other medicaments for preventing and/or treating advanced gastrointestinal stromal tumors located in the third container, or medicaments containing other medicaments for preventing and/or treating advanced gastrointestinal stromal tumors.

In another preferred embodiment, the kit further comprises (d 1) a fourth container, and a detection reagent for CDK1 in the fourth container.

In another preferred embodiment, the first container and the second container, the third container, and the fourth container are the same or different containers.

In another preferred embodiment, the medicament of the first container is a single formulation comprising an inhibitor of the CDK1 gene or protein thereof.

In another preferred embodiment, the medicament of the second container is a single formulation comprising a tyrosine kinase inhibitor.

In another preferred embodiment, the medicament of the third container is a single formulation containing other medicaments for preventing and/or treating advanced gastrointestinal stromal tumors.

In another preferred embodiment, the pharmaceutical is in the form of an oral dosage form or an injectable dosage form.

In another preferred embodiment, the kit further comprises instructions.

In another preferred embodiment, the instructions recite one or more instructions selected from the group consisting of:

(a) Use of an inhibitor of the CDK1 gene or protein thereof for (i) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) a method of increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors;

(b) Inhibitors of the CDK1 gene or protein thereof are used in combination with tyrosine kinase inhibitors, and/or optionally other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumor to (i) inhibit the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) increase resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors;

(c) Detecting the expression level of a CDK1 protein in a subject with advanced gastrointestinal stromal tumor, while administering an inhibitor of the CDK1 gene or protein thereof to (i) inhibit the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) a method of increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors;

(d) Detecting the expression level of CDK1 protein in a patient with advanced gastrointestinal stromal tumor, in combination with an inhibitor of CDK1 gene or protein thereof; and tyrosine kinase inhibitors, and/or optionally other agents for preventing and/or treating advanced gastrointestinal stromal tumor to (i) inhibit the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) methods of increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

The ninth aspect of the present invention provides the use of a pharmaceutical composition according to the sixth aspect of the present invention or a combination of products according to the seventh aspect of the present invention or a kit according to the eighth aspect of the present invention for the preparation of (i) a medicament for inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) a drug that increases the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

In another preferred embodiment, the concentration of inhibitor of the CDK1 gene or protein thereof in the pharmaceutical composition is in the range of from 100 to 3000ng/ml, preferably from 200 to 1000ng/ml, more preferably from 350 to 500ng/ml.

In another preferred embodiment, the concentration of the tyrosine kinase inhibitor in the pharmaceutical composition is in the range of 10-100000ng/ml, preferably 100-10000ng/ml, more preferably 500-2000ng/ml.

In another preferred embodiment, the concentration of the other drug for preventing and/or treating advanced gastrointestinal stromal tumor is between 10 and 100000ng/ml, preferably between 100 and 10000ng/ml, more preferably between 500 and 2000ng/ml.

In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of the CDK1 gene or protein thereof; and (b) optionally, a tyrosine kinase inhibitor; and (c) optionally other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumors; and (d) a pharmaceutically acceptable carrier.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof in the pharmaceutical composition or kit; and (b) optionally, a tyrosine kinase inhibitor; and (c) optionally, other agents for preventing and/or treating advanced gastrointestinal stromal tumors comprise 0.01-99.99wt%, preferably 0.1-90wt%, more preferably 1-80wt%, of the total weight of the pharmaceutical composition or kit.

In a tenth aspect the present invention provides a method of preventing and/or treating advanced gastrointestinal stromal tumors comprising:

Administering to a subject in need thereof an inhibitor of the CDK1 gene or protein thereof; or a pharmaceutical composition according to the sixth aspect of the invention or a product combination according to the seventh aspect of the invention or a kit according to the eighth aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal having advanced gastrointestinal stromal tumors.

In another preferred embodiment, the non-human mammal comprises a rodent and primate, preferably a mouse, rat, rabbit, monkey.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered at a dose of 0.5-10 mg/kg body weight, preferably 1-6mg/kg body weight, most preferably 3-5mg/kg body weight.

In another preferred embodiment, the tyrosine kinase inhibitor is administered at a dose of 1-600mg/kg body weight, preferably 2-60mg/kg body weight, most preferably 3-10mg/kg body weight.

In another preferred embodiment, the other agent for preventing and/or treating advanced gastrointestinal stromal tumors is administered at a dose of 0.06-600mg/kg body weight, preferably 1-60mg/kg body weight, most preferably 3-12mg/kg body weight.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered at a frequency of 2 to 5, preferably 3 to 4, times per week.

In another preferred embodiment, the tyrosine kinase inhibitor is administered at a frequency of 5 to 20 times per week, preferably 10 to 15 times per week.

In another preferred embodiment, the frequency of administration of the other agent for the prevention and/or treatment of advanced gastrointestinal stromal tumors is 3-15 times per week, preferably 6-10 times per week.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered for a period of 20 to 90 days, preferably 20 to 60 days, most preferably 30 to 40 days.

In another preferred embodiment, the tyrosine kinase inhibitor is administered for a period of 20 to 90 days, preferably 20 to 60 days, most preferably 30 to 40 days.

In another preferred embodiment, the additional agent for preventing and/or treating advanced gastrointestinal stromal tumors is administered for a period of 20-90 days, preferably 20-60 days, most preferably 30-40 days.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered simultaneously or sequentially with an optional tyrosine kinase inhibitor, and optionally with other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumors.

In an eleventh aspect, the invention provides a method of non-therapeutically inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells in vitro comprising the steps of: in the presence of CDK1 gene or its protein inhibitor, culturing the advanced gastrointestinal stromal tumor cells, thereby inhibiting the growth or proliferation of the gastrointestinal stromal tumor cells.

In another preferred embodiment, the CDK1 gene or protein inhibitor thereof is selected from the group consisting of: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof.

In another preferred embodiment, the advanced gastrointestinal stromal cells highly express a CDK1 protein.

In another preferred embodiment, the method further comprises adding a tyrosine kinase inhibitor to the culture system of advanced gastrointestinal stromal tumor cells; and/or other agents that prevent and/or treat advanced gastrointestinal stromal tumors, thereby inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells.

In another preferred embodiment, the advanced gastrointestinal stromal cells are cells cultured in vitro.

In a twelfth aspect, the present invention provides a method of screening for candidate compounds for the prevention and/or treatment of advanced gastrointestinal stromal tumors, the method comprising the steps of:

(a) In a test group, adding a test compound to a culture system of cells, and observing the expression level (E1) and/or activity (A1) of CDK1 in the cells of the test group; in the control group, no test compound was added to the culture system of the same cells, and the expression amount (E0) and/or activity (A0) of CDK1 in the cells of the control group was observed;

wherein, if the expression level (E1) and/or activity (A1) of CDK1 in the cells in the test group is significantly lower than that in the control group, the test compound is indicated to be a candidate compound for preventing and/or treating advanced gastrointestinal stromal tumor with an inhibitory effect on the expression and/or activity of CDK 1.

In another preferred embodiment, the CDK1 expression level is determined by fluorescent quantitative PCR or immunohistochemical or immunoblotting detection.

In another preferred embodiment, the method further comprises the steps of:

(b) Further testing the candidate compound obtained in step (a) for its inhibitory effect on the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or further testing for down-regulating effects on the CDK1 gene.

In another preferred embodiment, step (b) includes the steps of: in the test group, a test compound is added into a culture system of the advanced gastrointestinal stromal tumor cells, and the number and/or growth condition of the advanced gastrointestinal stromal tumor cells are observed; in the control group, no test compound is added in the culture system of the advanced gastrointestinal stromal tumor cells, and the number and/or growth condition of the advanced gastrointestinal stromal tumor cells are observed; wherein, if the number or growth rate of the advanced gastrointestinal stromal tumor cells in the test group is smaller than that of the control group, the test compound is indicated to be a candidate compound for preventing and/or treating the advanced gastrointestinal stromal tumor with an inhibitory effect on the growth or proliferation of the advanced gastrointestinal stromal tumor cells.

In another preferred embodiment, the method comprises step (c): administering the candidate compound determined in step (a) to a mammalian model, and determining its effect on the mammal.

In another preferred embodiment, the mammal is a mammal having an advanced gastrointestinal stromal tumor.

In another preferred embodiment, the term "substantially lower" means E1/E0.ltoreq.1/2, preferably.ltoreq.1/3, more preferably.ltoreq.1/4.

In another preferred embodiment, the term "significantly lower" means A1/A0.ltoreq.1/2, preferably.ltoreq.1/3, more preferably.ltoreq.1/4.

In another preferred embodiment, the cells comprise advanced gastrointestinal stromal tumor cells.

In another preferred embodiment, the cells are cells cultured in vitro.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows (A) a volcanic plot of RNA-seq showing the pattern of differential gene expression between early and late GISTs. (B) Pattern drawing of end-stage GIST cell whole genome CRISPR screening. The box plot (C) shows the distribution of sgRNA at various doublings. (D) In the whole genome CRISPR screen of GIST430/654 cells, the CRISPR score of each gene was ranked. CDK1 ranks second. All genes analyzed in the CRISPR screen are identified in gray, and 568 genes identified in whole transcriptome sequencing that are highly expressed in late GIST are identified in black.

Fig. 2 shows (a) the expression profile of CDK1, CDK4, CDK6 and CDK9 in the GIST discovery cohort (early GIST n=11, late n=32), CDK1 being significantly more highly expressed in late GIST than in early GIST compared to other CDKs. (B) Once CDK1 is highly expressed in one metastasis, it is highly expressed in all other metastases of the same GIST patient. (C) Ki67 is a proliferative molecular marker, CDK1 and Ki67 expression are highly correlated (D) western blots (n=92) showing no/low expression of CDK1 in early GIST but frequent expression in late GIST. (E) Immunohistochemical detection in formalin fixed paraffin embedded GIST clinical samples showed high CDK1 expression in late GIST, whereas CDK1 expression levels in early GIST were low. (F) Tissue chip sample statistical analysis containing 325 early GIST and 177 late GIST. High expression of CDK1 is highly correlated with malignancy of GIST.

FIG. 3 shows (A) CDK1 knockdown in imatinib resistant GIST430/654 and imatinib sensitive GIST-T1 cell lines detected by qRT-PCR. (B) Lentiviral mediated CDK1 knockdown significantly reduced the viability of GIST430/654 and GIST-T1 cells in the short term, as assessed by the CellTiter-Glo viability assay. (C) Crystal violet staining showed that CDK1 knockdown inhibited GIST cell proliferation for long periods. (D) CDK1 knockdown inhibited the anchorage-independent growth of GIST430/654 and GIST-T1 cells. (E) CDK1 knockdown inhibited tumor growth in GIST430/654 and GIST-T1 xenograft mice. (F) Cell cycle analysis showed that CDK1 knockdown reduced the proportion of cells in the S phase and G2/M phase of GIST430/654 and GIST-T1, and increased the proportion of cells in the G0/G1 phase, resulting in G0/G1 arrest. (G) Cell senescence staining analysis showed that CDK1 knockdown significantly increased the proportion of senescent cells of GIST430/654 and GIST-T1, promoting cell senescence, which was also one of the causes of G0/G1 cell cycle arrest.

Fig. 4 shows (a) identification of proteins interacting with CDK1 by co-immunoprecipitation and mass spectrometry-based proteomics. GIST430/654 cells were transduced with FLAG-CDK1 lentivirus and a pulldown assay was performed using FLAG antibodies. The table shows the high confidence proteins in the mass spectrum, AKT1 rank second. (B) AKT-CDK1 interactions in PDK1 normal and PDK1 deficient GIST430/654 cells were validated. GIST430/654 cells were transduced with FLAG-CDK1 and cell lysates were subjected to co-immunoprecipitation analysis using FLAG antibodies, followed by immunoblotting using AKT antibodies, indicating that CDK1 and AKT binding was independent of PDK1. (C) Treatment with the inhibitor MK-2206 of AKT inhibited CDK1 binding to AKT and reduced the level of phosphorylation of AKT in PDK1 normal GIST430/654 cells. (D) Endogenous CDK1 interacts strongly with AKT in the GIST430/654 cell line. Cell CDK1 and AKT IP immunoblots treated with control or CDK1 inhibitors (RO-3306,1. Mu.M). Left, cell lysates were immunoprecipitated with CDK1 antibody and immunoblotted with AKT antibody. On the right, cell lysates were immunoprecipitated with AKT antibody and immunoblotted with CDK1 antibody. It was shown that AKT1 interacts with CDK1 in the GIST system. (E) In vitro interaction analysis also showed that CDK1 interacted with AKT and that this interaction was attenuated upon inhibition by RO-3306. . (F) In 293 cells, co-immunoprecipitation showed strong interaction between CDK1 and AKT1 after exogenous introduction of FLAG-CDK1 and HA-AKT1, and CDK1 promoted phosphorylation of threonine at AKT 308 and serine at 473. (G) In PDK1 knocked-out GIST430/654 cells, after exogenous introduction of FLAG-CDK1 and HA-AKT1, co-immunoprecipitation showed strong interaction of CDK1 and AKT1 and promoted phosphorylation of threonine at AKT 308 and serine at 473. It was shown that CDK1 may activate AKT alone independently of PDK1. (H) Knocking down CDK1 in GIST430/654 and GIST-T1 cells inhibited AKT phosphorylation, induced apoptosis and reduced PCNA expression. (I) Treatment of GIST430/654 and GIST-T1 cells with RO-3306 inhibited AKT phosphorylation, induced apoptosis, and inhibited cell proliferation as did CDK1 knockdown.

FIG. 5 shows that (A) the phenotype of inhibiting cell viability after CDK1 knockdown was complemented by the addition of AKT after CDK1 knockdown in GIST 430/654. GIST430/654 cell viability was assessed by CellTiter-Glo viability assay. (B) After CDK1 knockdown in GIST430/654, AKT was supplemented back, complementing the phenotype of inhibiting cell proliferation after CDK1 knockdown. GIST430/654 cell proliferation was assessed by crystal violet staining assay. (C) AKT was supplemented back after CDK1 knockdown in GIST430/654, compensating for the phenotype of inhibiting cell anchorage independence after CDK1 knockdown. GIST430/654 cell anchorage independent growth was assessed by soft agar experiments. (D) AKT was supplemented back after CDK1 knockdown in GIST430/654, compensating for tumor growth in xenograft mice following CDK1 knockdown. CDK1 was shown to promote GIST cell growth and proliferation by AKT.

FIG. 6 shows (A) CellTiter-Glo growth inhibition assay for CDK1 inhibitor RO-3306 in GIST cell lines and GIST primary cells. GIST-1, BJ, was established from a metastatic GIST without CDK1 expression, a non-transformed fibroblast cell line. RO-3306 inhibited imatinib-sensitive and drug-resistant GIST cell lines, without affecting GIST primary and fibroblasts that did not express CDK 1. (B) Half inhibition concentration (IC 50) of RO-3306 on imatinib resistant and imatinib sensitive GIST cells. (C) Immunoblots showed expression levels of CDK1 in GIST cells and GIST primary cells. (D) Immunoblots showed that RO-3306 treated imatinib resistant and imatinib sensitive GIST cells did not affect AKT expression, but significantly reduced AKT phosphorylation levels. Anti-tumor activity of (E-G) CDK1 inhibitors against imatinib-resistant GIST xenograft mice. Treatment of imatinib resistant patient-derived xenograft GIST tumor mice with CDK1 inhibitors (ex 11+ ex 17) patient-derived xenograft GIST tumor-bearing mice were treated with 4mg/kg RO-3306 per 2 days or 50mg/kg imatinib twice daily, reducing tumor volume as well as tumor size. And CDK1 inhibitors did not affect body weight changes in imatinib-resistant GIST xenograft mice. (H) The combination of RO-3306 and imatinib treated GIST-T1 cells significantly inhibited GIST cell growth than treatment alone. (I-K) anti-tumor Activity of RO-3306 in GIST xenograft mice sensitive to imatinib. Mice bearing GIST-T1 tumors were orally administered twice daily as a single drug or in combination with 4mg/kg RO-3306 or a low dose (25 mg/kg) of imatinib every 2 days. The CDK1 inhibitor in combination with imatinib treated imatinib-sensitive GIST-T1 tumor-bearing mice reduced tumor volume as well as tumor size. And CDK1 inhibitors did not affect body weight changes in imatinib-sensitive GIST xenograft mice. The CDK1 inhibitors were shown to have anti-tumor activity against imatinib resistant and imatinib sensitive late GIST.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, unexpectedly found that the expression of CDK1 gene or protein thereof in advanced gastrointestinal stromal tumor cells or tissues is significantly higher than the expression of CDK1 gene or protein thereof in early gastrointestinal stromal tumor tissues, and thus, CDK1 gene or protein thereof can be used as a marker for detecting advanced gastrointestinal stromal tumors. Furthermore, the applicant has unexpectedly found that inhibitors of the CDK1 gene or protein thereof are effective in (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increase the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors, and inhibitors of the CDK1 gene or protein thereof may be used in combination with tyrosine kinase inhibitors, and/or optionally other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumors, and have a significant synergistic effect on the treatment of advanced gastrointestinal stromal tumors. On this basis, the present inventors have completed the present invention.

As used herein, the term "RO-3306" has the formula

NU6027 has the structural formula of

Flavopiridol has the structural formula

AT7519 has the structural formula of

GW5074 has the structure of/>

Dinaciclib has the structural formula

The structural formula of JNJ-7706621 is

AZD5438 has the structural formula

BMS-265246 has the structural formula of

R547 has the structural formula

CDKI-73 has the structural formula

Alsterpaullone has the structural formula of

SU9516 has the structural formula of/>

Avapritinib (BLU-285) has the structural formula

The structural formula of the DCC-2618 is

Gastrointestinal stromal tumor

Gastrointestinal stromal tumors are the most common gastrointestinal stromal She Yuanxing tumors, clinically symptomatic gastrointestinal stromal tumors are found in adults over 45 years of age, and gastrointestinal stromal tumors can occur throughout the digestive tract, but most occur in the stomach and small intestine, with clinical stromal tumors being the most common sarcoma and having a annual incidence of 10-20/100 ten thousand people. With advances in endoscopic and imaging techniques, more and more small-volume gastrointestinal stromal tumors are found. Many pathological studies have demonstrated that small gastrointestinal stromal tumors with diameters less than 1cm are common in middle-aged and elderly people, with a discovery rate of up to 35%, and with the aggravation of the aging world population, it is estimated that small gastrointestinal stromal tumor patients in China reach 1 million people.

The primary pathogenesis of gastrointestinal stromal tumors is the activation mutation of the protooncogene KIT in kahal stromal cells, thereby abnormally activating downstream signaling pathways, mainly including the MAPK pathway and PI3K-AKT pathway, such that cell survival, growth and proliferation are uncontrolled.

Advanced gastrointestinal stromal tumor

Gastrointestinal stromal tumors are classified into primary gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors according to the presence or absence of metastasis, and primary gastrointestinal stromal tumors are classified into low-risk, medium-risk and high-risk gastrointestinal stromal tumors according to pathological indexes (tumor size, mitosis per high-power visual field, anatomical position, and the like), for example, high-risk gastrointestinal stromal tumors refer to a high risk of metastasis and recurrence. High-risk gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors are collectively referred to as advanced gastrointestinal stromal tumors. The prognosis of the advanced gastrointestinal stromal tumor is poor, and the clinic needs a treatment means urgently. About 80% of gastrointestinal stromal tumors contain KIT activating mutations, molecular targeted therapy targeting KIT oncoproteins innovates the therapeutic regimen for stromal tumors during progression, but molecular heterogeneity among individuals of gastrointestinal stromal tumors leads to a high degree of inconsistency in the response of different individuals to targeted therapy. The gene detection plays an important role in predicting the curative effect of gastrointestinal stromal tumor targeted therapy, disease prognosis and the like. At present, for gastrointestinal stromal tumor patients receiving targeted therapy, almost all patients develop drug resistance, and how to improve the therapeutic effect of targeted therapeutic drugs represented by imatinib is an urgent problem to be solved in clinical medicine and basic medicine.

Sample of

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject. The sample may also be a material that has been contacted with the subject in a manner that allows the test performed on the sample to provide information about the subject. The sample may also be a material that has been contacted with another material that is not the subject, but that enables the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a cleaning solution for a probe or scalpel. The sample may be a source of biological material other than that contacting the subject, so long as one skilled in the art is still able to determine information about the subject from the sample.

Expression of

As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density in immunoblots, such as western blots, is also within the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically relevant to a particular result when compared to the result of an analysis. In a preferred embodiment, the reference value is determined from a statistical analysis performed in conjunction with studies comparing the expression of CDK1 protein with known clinical results. Some of these studies are shown in the examples section herein. But the studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. Reference values may also be determined by considering conditions and results that are particularly relevant to the patient's medical history, genetics, age and other factors.

In the present invention, the reference value refers to a cut-off value, which refers to the relative expression level of CDK1 in cells or tissues of advanced gastrointestinal stromal tumor, preferably a relative expression level of 5 (FPKM value, FPKM: FRAGMENTS PER Kilobase of exon model per Million MAPPED FRAGMENTS, fragments read per million maps per kilobase of transcription, is obtained by transcriptome sequencing), from a certain level of RNA expression

Samples of non-advanced gastrointestinal stromal tumors

As used herein, the term "non-advanced gastrointestinal stromal tumor sample" includes, but is not limited to, a population not having advanced gastrointestinal stromal tumor, non-advanced gastrointestinal stromal tumor tissue of an advanced gastrointestinal stromal tumor patient.

CDK1 proteins and polynucleotides

In the present invention, the terms "protein of the invention", "CDK1 protein", "CDK1 polypeptide" are used interchangeably and refer to a protein or polypeptide having a CDK1 amino acid sequence. They include CDK1 proteins with or without an initiating methionine. Furthermore, the term also includes full length CDK1 and fragments thereof. The CDK1 proteins referred to in the present invention include their complete amino acid sequences, their secreted proteins, their mutants and functionally active fragments thereof.

CDK1 is cyclin dependent kinase 1, which acts as a serine/threonine kinase and is a key player in cell cycle regulation. In humans, CDK1 is encoded by the CDC2 gene. It is essential for G1/S and G2/M phase changes in eukaryotic cell cycles. CDK1 forms a complex with cyclin that phosphorylates a variety of target substrates (more than 75 have been identified in germinated yeast). The phosphorylation and dephosphorylation of this protein plays an important regulatory role in cell cycle control.

The human CDK1 protein is 297 amino acids in length (accession NP-001777). The murine CDK1 protein is 297 amino acids in full length (accession NP-031685).

In the present invention, the terms "CDK1 gene", "CDK1 polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a CDK1 nucleotide sequence.

The genome of the human CDK1 gene is 23522bp (NCBI GenBank accession number NG_ 029877.1), and the mRNA sequence of the transcription product is 1889bp (NCBI GenBank accession number NM_ 001786).

The genome of the murine CDK1 gene is 17767bp in length (NCBI GenBank accession NC-000076.7), and the transcript mRNA sequence is 4362bp in length (NCBI GenBank accession NM-007659.4).

Human and murine CDK1 showed a DNA level of 76.25% similarity and a protein sequence similarity of 96.97%.

It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the case where an amino acid fragment of CDK1 is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For PCR amplification, primers may be designed according to the CDK1 nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified to give the relevant sequences using a commercially available cDNA library or cDNA library prepared by conventional methods known to those skilled in the art as templates. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the invention (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the invention may be used to express or produce recombinant CDK1 polypeptides by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human CDK1 polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, the CDK1 polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing CDK1 encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells; animal cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Specific antibodies

In the present invention, the terms "antibody of the invention" and "specific antibody against CDK 1" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for a human CDK1 polypeptide. Herein, "specific" means that the antibody is capable of binding to a human CDK1 gene product or fragment. Preferably, those antibodies which bind to a human CDK1 gene product or fragment but which do not recognize and bind to other non-related antigenic molecules. Antibodies in the present invention include those molecules capable of binding to and inhibiting the human CDK1 protein, as well as those antibodies that do not affect the function of the human CDK1 protein. The invention also includes antibodies that bind to the modified or unmodified form of the human CDK1 gene product.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments such as the Fab' or (Fab) ₂ fragments; antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, a purified human CDK1 gene product, or an antigenic fragment thereof, may be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing the human CDK1 protein or antigenic fragment thereof may be used to immunize animals to produce antibodies. The antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al, nature 256;495,1975; kohler et al, eur. J. Immunol.6:511,1976; kohler et al, eur. J. Immunol.6:292, 1976; hammerling et al, inMonoclonalAntibodiesandTCellHybridomas, elsevier, N.Y., 1981). Antibodies of the invention include antibodies that block the function of human CDK1 protein and do not affect the function of human CDK1 protein. The various antibodies of the invention may be obtained by conventional immunological techniques using fragments or functional regions of the human CDK1 gene product. These fragments or functional regions may be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind to an unmodified form of a human CDK1 gene product may be produced by immunizing an animal with the gene product produced in a prokaryotic cell (e.g., e.coli); antibodies (e.g., glycosylated or phosphorylated proteins or polypeptides) that bind to post-translational modifications can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

Antibodies against human CDK1 protein are useful in immunohistochemical techniques to detect the presence of human CDK1 protein in a sample, particularly a tissue sample or serum sample.

Detection method

The invention also provides a method for detecting the advanced gastrointestinal stromal tumor by utilizing the characteristic that CDK1 is highly expressed in cells or tissues of the advanced gastrointestinal stromal tumor.

In a preferred embodiment of the invention, the invention provides a high throughput second generation sequencing method for detecting CDK1 as well as Sanger sequencing, fluorescent quantitative PCR (qPCR), immunoblotting, in situ immunofluorescence (FISH), immunohistochemistry and the like.

Detection kit

Based on the correlation of CDK1 with advanced gastrointestinal stromal tumor, i.e., CDK1 is present in advanced gastrointestinal stromal tumor tissue, CDK1 can be a diagnostic marker for advanced gastrointestinal stromal tumor.

The invention also provides a kit for detecting advanced gastrointestinal stromal tumor, which contains a detection reagent for detecting CDK1 genes, mRNA, cDNA or protein; and a label or instructions stating that the kit is for detecting advanced gastrointestinal stromal tumors.

Wherein the label or instructions note that the kit is used for detecting advanced gastrointestinal stromal tumors.

Detection method and kit

The present invention relates to diagnostic assays for the quantitative and positional detection of human CDK1 protein levels or mRNA levels. Such tests are well known in the art. The levels of human CDK1 protein detected in the assay may be used to diagnose (including aid in diagnosis of) advanced gastrointestinal stromal tumors.

A method of detecting the presence or absence of a CDK1 protein in a sample using an antibody specific for a CDK1 protein comprising: contacting the sample with an antibody specific for CDK1 protein; whether an antibody complex is formed is observed, and the formation of an antibody complex indicates the presence of CDK1 protein in the sample.

The CDK1 protein or polynucleotide thereof may be used in the diagnosis and treatment of CDK1 protein-related disorders. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in tissues and diagnosis of genes. Antibodies against CDK1 may be immobilized on a protein chip for detection of CDK1 proteins in a sample.

The main advantages of the invention include:

(1) The present invention for the first time found that the expression of CDK1 gene or protein thereof in advanced gastrointestinal stromal tumor cells or tissues is significantly higher than that of CDK1 gene or protein thereof in normal tissues, and thus CDK1 gene or protein thereof can be used as a marker for detecting advanced gastrointestinal stromal tumors.

(2) The present invention for the first time has found that inhibitors of the CDK1 gene or protein thereof are effective in (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increase the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

(3) The present invention for the first time has found that inhibitors of the CDK1 gene or protein thereof may be used in combination with tyrosine kinase inhibitors, and/or optionally other agents for the prophylaxis and/or treatment of advanced gastrointestinal stromal tumors, and have a significant synergistic effect in the treatment of advanced gastrointestinal stromal tumors.

(4) The present invention for the first time found that CDK1 is highly expressed in tissue samples from patients with advanced gastrointestinal stromal tumors.

(5) The present invention for the first time found that CDK1 promotes the growth and proliferation of gastrointestinal stromal tumor cells.

(6) The present invention for the first time has found that inhibiting the expression or activity of CDK1 can inhibit the growth and proliferation of advanced gastrointestinal stromal tumors (GIST).

(7) The present invention for the first time has found that inhibiting CDK1 expression or activity can significantly increase GIST's resistance and sensitivity to tyrosine kinase inhibitors (such as imatinib).

(8) The present invention for the first time found that CDK1 promotes GIST growth and proliferation through AKT.

(9) The present invention for the first time found that CDK1 binds and modulates AKT phosphorylation.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.

Example 1 Whole genome CRISPR (clustered regularly interspaced short palindromic repeats) screening found that CDK1 was the target of advanced gastrointestinal stromal tumor (GIST)

To find functional genes that affect GIST cells, the present study uses CRISPR/Cas9 to inactivate screen GIST430/654 cells. GIST430/654 cells were infected with GeCKOv library virus at an efficiency of approximately 0.4 in MOI, and all cells were divided into 2 groups after puromycin (puromycin) screening, with the number of cells per group not less than 300 x library coverage. The first group extracted cell genomic DNA served as Day 0 control, and the second group was cultured for 15 passages continuously and then extracted. The genomic DNA was subjected to nested PCR to amplify the introduced sgRNA and to introduce barcodes sequences, and the amplicons were subjected to second generation sequencing with a depth of more than 300X. Based on the change in sgRNA reading during CRISPR/Cas9 inactivation screening, CRISPR GENE score (CS) was calculated and used to reflect the importance of the gene.

The experimental steps are as follows:

1) Transcriptome data from 43 GIST samples were analyzed (fig. 2A-C);

2) Whole genome CRISPR screening using an advanced GIST cell line (GIST 430/654) to knock out genes that inhibit cell proliferation;

3) The transcriptome data and CRISPR screening data were used in combination to screen for weaknesses in late GIST (fig. 1A-D).

The experimental results are shown in fig. 1 a-D.

The results showed that 568 genes were highly expressed in late GIST and low in early GIST (fig. 1A); GIST430/654 cells were screened for late imatinib resistance using the method CRISPR SCREEN of FIG. 1B; after 15 Population Doublings (PD), the distribution of sgrnas was significantly altered compared to PD0 cells, suggesting that the functional screen was designed (fig. 1C); in the GIST430/654 screening results, the CS values of CDK1 were ranked second (fig. 1D). CDK1 was generally shown to be a potential target for advanced gastrointestinal stromal tumors (GISTs).

Example 2 significantly high expression of CDK1 in late GIST

Immunoblotting experiments: IP buffer was added to the harvested sample or cellular protein, after 4℃overnight lysis, the protein was quantified by centrifugation at 12000rpm for 30min using Quick Start ^TM Bradford 1X DYE REAGENT (Bio-Rad; # 5000205). Electrophoresis and western blotting were performed using standard techniques. Hybridization signals were detected by chemiluminescence (Immobilon Western, mi llipore Corporation, MA) and captured using a AMERSHAM IMAGER imager (GE HEALTHCARE; # 29083461).

Immunohistochemistry: immunohistochemistry was performed on tissues and tumor sections using CDK1 antibody (Santa Cruz #sc-54). Xylene was used for dewaxing and washed in a series of ethanol of different concentrations. Slides were boiled in citrate buffer (pH 6) by microwaves for 12 minutes. Immunohistochemical reactions were observed by diaminobenzidine staining.

The experimental steps are as follows:

1) CDK1 expression levels were detected by Western blotting in early and late GISTs (FIG. 2D);

2) CDK1 expression levels were detected by immunohistochemistry in early and late GIST (fig. 2E);

3) CDK1 expression levels were detected by tissue chip samples of 502 GIST (fig. 2F).

The experimental results are shown in FIG. 2 (A-F).

The results indicated that the 43 GIST sample RNA-seq results showed that only CDK1 was highly expressed (RNA level) in late GIST relative to other CDKs (fig. 2A); CDK1 was found to be highly expressed in one metastasis of one patient, and CDK1 was found to be highly expressed in the remaining metastasis (FIG. 2B); CDK1 expression was significantly positively correlated with KI67 expression (fig. 2C); the 92 samples immunoblotted results showed that the ratio of expression levels in the late GIST was 38/52 (72%) significantly higher than the early GIST (protein levels) (fig. 2D); 502 TMA immunohistochemical chips showed that CDK1 high expression was significantly correlated with early and late stages of GIST, with high expression in late GIST (FIG. 2, E-F). All 3 queues showed significantly high expression of CDK1 in late GIST.

EXAMPLE 3 CDK1 Gene deletion inhibits GIST growth and proliferation

Lentivirus packaging: transfection of 293T cells was mediated with polyethylenimine (polyethylenime, PEI). Cells were plated at appropriate densities into 10cm dishes one day before transfection, and the cells were plated with serum-free medium at about 70-90% growth, and after 2 hours the plasmid was transfected with PEI. The lentiviral packaging plasmids were 9. Mu.g.delta.8.9 and 3.5. Mu. g vsv-g, the plasmid of interest 10. Mu.g. The complete culture medium is used for liquid exchange 4-6 hours after transfection, and the supernatant is collected after 24, 36, 48 and 60 hours to obtain virus liquid.

Cell viability detection: detection was performed using CellTiter-Glo (CTG) kit. The treated 96-well plate cells are incubated for 30 minutes at room temperature, CTG reagent is diluted 4 times by PBS, 100 mu L of diluted CTG reagent is added to each well of the cells, the cells are placed on a shaking table for mixing for 2 minutes at room temperature and in a dark condition to lyse the cells, and the cells are kept stand for 10 minutes and then the luminous intensity is detected by an enzyme-labeled instrument.

Crystal violet experiment: after cells in the 6-well plate grew to a certain extent, the medium was poured off, washed twice with PBS, and fixed by adding 4% paraformaldehyde to each well for 20min. After repeated PBS washes, the cells were incubated for 30min at room temperature in the dark using crystal violet staining solution. Washing with flowing water for several times, and taking photos under an optical microscope for counting.

Soft agar experiments: after the 6-well plate cells grew to a certain extent, the medium was poured out, 1X MTT staining solution was added, and the mixture was placed in a 5% CO2 incubator. After 2h, the clone was stained blue-violet, and the clone was subjected to photographing scanning under an optical microscope.

Cell cycle detection: the cultured cells are prepared into single cell suspension, the single cell suspension is fixed by 75% ethanol at-20 ℃ after PBS washing, the single cell suspension is washed by PBS after 24 hours, RNase A is added, the single cell suspension is evenly mixed and incubated at 37 ℃ for 30 minutes, propidium Iodide (PI) is added for dyeing DNA, and the single cell suspension is detected by a flow cytometer after incubation at room temperature for 30 minutes in a dark place.

Nude mice xenograft experiments: the obtained GIST cells and matrigel were uniformly mixed in a ratio of 1:1, and inoculated subcutaneously in the armpit of BALB/c nude mice using a syringe. Mice were observed daily for physiological status and tumor size was weighed. For drug-treated mice, once the tumor had grown to a certain extent, the dose of imatinib was twice daily, 50mg/kg, and the dose of RO-3306 was 4mg/kg every two days, and the stomach was irrigated orally for 30 days. After 30 days of treatment, all mice were sacrificed and tumors were collected. Tumor volume and tumor weight were used to evaluate anti-tumor activity.

SA-beta-Gal cell aging detection experiment: cells after lentivirus infection were cultured until a clear cell senescence state was seen under a microscope, medium in the well plate was discarded, washed twice with PBS, and fixed with 4% paraformaldehyde for 20min. The incubation was performed twice with running water and stained overnight with the beta-galactosidase activity detection kit from Biyundin. Discarding staining reagent, observing under an optical microscope, taking photos of random 3 fields, artificially counting stained cells, and counting aging percentage.

The experimental steps are as follows:

1) Constructing a CDK1 gene lentivirus knockdown vector;

2) Infection of GIST430/654 and GIST-T1 cells after packaging lentiviruses;

3) Cell viability was measured using CellTiter-Glo (CTG) kit (FIGS. 3A-B);

4) Immunoblots detected CDK1 knockdown levels (fig. 4H);

5) Proliferation ability of CDK1 knockdown cells was tested by crystal violet, soft agar experiments (FIGS. 3C-D);

6) Flow cytometry detects the proportion of each phase of the cell cycle, reflecting the proliferation of the cells (fig. 3F);

7) Injecting cells into nude mice subcutaneously for xenograft, and periodically observing and detecting tumor growth (fig. 3E);

8) Cell senescence kits detect the senescence level of cells following CDK1 knockdown (fig. 3G).

The experimental results are shown in FIG. 3 (A-G).

The results showed that after CDK1 knockdown, cell viability experiments, cell anchorage independent experiments, crystal violet experiments, nude mice transplantation experiments all showed inhibition of GIST cell proliferation (FIG. 3, A-E); CDK1 knockdown was followed by cell cycle arrest at G0/G1 (FIG. 3, F) due to promotion of cell senescence (FIG. 3, G). Thus, CDK1 gene knockout significantly inhibited GIST cell growth and proliferation, promoting GIST senescence.

Example 4 CDK1 binds to and modulates phosphorylation of AKT

And (3) constructing a carrier: full-length AKT and FLAG-CDK1 were amplified by molecular cloning and ligated into expression vectors by the cleavage ligation method.

Immunoprecipitation mass spectrometry: cells were lysed with IP buffer containing protease inhibitor (10. Mu.g/mL leupeptin, 10. Mu.g/mL aprotinin and 1mM phenylmethylsulfonyl fluoride), mixed overnight with 1. Mu.g anti-FLAG antibody and 20. Mu.L protein G-agarose (ThermoFisher # 101242) and eluted by boiling in SDS loading buffer. Eluted samples were detected by SDS-PAGE, coomassie blue staining (colloidal blue staining kit, invitrogen, # LC 6025). For mass spectrometry, IP samples were eluted by shaking with 8M urea and 100mM Tris-Cl (pH 8.0) and mass spectrometry was performed.

Co-immunoprecipitation: cells were lysed with IP buffer containing protease inhibitors (10. Mu.g/mL leupeptin, 10. Mu.g/mL aprotinin and 1mM phenylmethylsulfonyl fluoride), mixed with 2. Mu.g of anti-Flag antibody and 20. Mu.L of protein G-agarose and incubated overnight. Immunoprecipitates were eluted by boiling with SDS loading buffer. IP samples and whole cell lysates were analyzed by western blot.

The experimental steps are as follows:

1) Constructing a FLAG-CDK1 exogenous expression vector;

2) The FLAG-CDK1 was overexpressed in GIST430/654 and the CDK1 interacting proteins were pulled down using FLAG antibodies, mass spectrometry identified (FIG. 4A);

3) FLAG-CDK1 was overexpressed in GIST430/654 and the interaction protein AKT was validated using FLAG antibody pull down (fig. 4D);

4) Treatment with CDK1 inhibitor RO-3306 in GIST430/654, demonstrated CDK 1-specific interaction protein AKT (FIG. 4D);

5) In an in vitro binding assay, CDK1 inhibitor RO-3306 was incubated and CDK1 antibody was used to pull down to verify CDK1 interacting protein AKT1 (fig. 4E);

6) In the 293 system, FLAG-CDK1 and HA-AKT were exogenously introduced, and CDK1 and AKT interactions were verified using FLAG antibody pull down (FIG. 4F);

7) Exogenous FLAG-CDK1 was introduced into PDK1 normal and defective GIST430/654 cells, and by FLAG antibody pulldown, it was verified that CDK1 interaction with AKT was independent of PDK1 (FIG. 4B);

8) In PDK1 normal GIST430/654, cells were treated with the AKT inhibitor MK-2206, pulled down with FLAG antibody, CDK1 binding to AKT was verified, immunoblotted with whole cell lysates, showing that inhibitor treated groups reduced phosphorylation of AKT (FIG. 4C);

9) In PDK1 knocked-out GIST430/654 cells, exogenously introduced FLAG-CDK1 and HA-AKT1, the interacting protein AKT was verified using FLAG pulldown, immunoblots showed reduced phosphorylation of AKT (FIG. 4G);

10 Knocking down CDK1 in GIST430/654 and GIST-T1 or detecting the expression levels of AKT/pAKT, PCNA and PARP, etc. by Western blotting using CDK1 inhibitor RO-3306 (FIG. 4H-I);

The experimental results are shown in FIG. 4 (A-I).

The results indicate that AKT1 ranks second among proteins pulled down using antibody FLAG in GIST430/654 (fig. 4A); the binding of CDK1 and AKT is independent of PDK1 (fig. 4B); treatment with an inhibitor of AKT MK-2206 inhibited CDK1 binding to AKT and reduced AKT phosphorylation levels in PDK1 normal GIST430/654 cells (fig. 4C); in GIST cell system AKT1 interacts with CDK1 (fig. 4D); in vitro interaction analysis also showed that CDK1 interacted with AKT (fig. 4E); in 293 cell systems, AKT1 interacts with CDK1, and CDK1 promotes phosphorylation of threonine at AKT 308 and serine at 473 (fig. 4F); knocking down CDK1 in GIST430/654 and GIST-T1 cells, inhibiting AKT phosphorylation, inducing apoptosis, and reducing PCNA expression (FIGS. 4G, H); treatment of GIST430/654 and GIST-T1 cells with RO-3306 inhibited AKT phosphorylation, induced apoptosis, and inhibited cell proliferation as with CDK1 knockdown (fig. 4I). Thus, CDK1 binds and modulates AKT phosphorylation, and is independent of PDK1, while CDK1 knockdown promotes apoptosis.

Example 5 CDK1 promotes GIST growth and proliferation by AKT

The experimental steps are as follows:

1) The AKT was supplemented back after CDK1 knockdown in GIST430/654 and GIST growth and proliferation were detected by CellTiter-Glo viability, crystal violet and soft agar experiments (fig. 5A-C);

2) In GIST430/654, AKT was supplemented back after CDK1 knockdown, and GIST growth and proliferation were examined by in vivo experiments such as nude mice transplantation (FIG. 5D).

The results indicate that the anaplerotic AKT complements the phenotype of inhibiting cell viability following CDK1 knockdown (fig. 5A); AKT was supplemented back to complement the phenotype of inhibition of cell proliferation following CDK1 knockdown (fig. 5B); AKT was supplemented back to complement the phenotype of inhibition of cell anchorage independence following CDK1 knockdown (fig. 5C); AKT was supplemented back to compensate for inhibition of tumor growth in xenograft mice following CDK1 knockdown (fig. 5D). Thus, CDK1 promotes GIST growth and proliferation through AKT.

Example 6 antitumor Activity of CDK1 inhibitors against Imatinib resistance and Imatinib-sensitive late GIST

The experimental steps are as follows:

1) 7 GIST cells were treated with gradient concentrations of RO-3306 and primary GIST cells, cell titer-Glo viability kit was used to measure cell viability and to count half inhibition concentrations (fig. 6A-B);

2) The CDK1 expression levels in GIST cells and GIST primary cells were shown by immunoblotting (fig. 6C);

3) Treatment of GIST cells with RO-3306 and GIST primary cells, immunoblots showed a decrease in AKT phosphorylation levels (fig. 6D);

4) In the GIST-T1 cell line, the combined treatment with RO-3306 and imatinib was significantly superior to monotherapy (fig. 6H);

5) Tumor size, volume and toxic effects on mice were tested for patient-derived xenograft mice (PDX) using RO-3306 or a combination of RO-3306 and imatinib (FIGS. 6E-G, I-K).

The results indicate that RO-3306 inhibited imatinib-sensitive and drug-resistant GIST cell lines, without affecting GIST primary and fibroblasts that CDK1 did not express (FIG. 6, A-B); expression levels of CDK1 in GIST cells and GIST primary cells (FIG. 6C); RO-3306 treated imatinib resistant and imatinib-sensitive GIST cells did not affect AKT expression, but significantly reduced AKT phosphorylation levels (FIG. 6D); CDK1 inhibitors inhibited tumor volume and size in imatinib-resistant GIST xenograft mice and were non-toxic (FIG. 6,E-G); the CDK1 inhibitor in combination with imatinib treated imatinib-sensitive GIST-T1 tumor-bearing mice reduced tumor volume as well as tumor size. And CDK1 inhibitors did not affect body weight changes in imatinib-sensitive GIST xenograft mice (fig. 6, i-K). Thus, CDK1 inhibitors have anti-tumor activity against imatinib resistance and imatinib-sensitive late GIST.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. Use of an inhibitor of a CDK1 gene or protein thereof, for the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors, wherein the inhibitor of the CDK1 gene or protein thereof is RO-3306.

2. A pharmaceutical composition for treating advanced gastrointestinal stromal tumors, comprising:

(a1) An inhibitor of the CDK1 gene or protein thereof, said inhibitor being RO-3306;

(a2) A tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is imatinib; and

(B) A pharmaceutically acceptable carrier.

3. A combination of products for the treatment of advanced gastrointestinal stromal tumors, comprising:

(i) A first pharmaceutical composition comprising (a) a first active ingredient which is an inhibitor of the CDK1 gene or protein thereof, said inhibitor being RO-3306, and a pharmaceutically acceptable carrier; and

(Ii) A second pharmaceutical composition comprising (b) a second active ingredient which is a tyrosine kinase inhibitor which is imatinib, and a pharmaceutically acceptable carrier.

4. A product combination according to claim 3, wherein the product combination further comprises a detection reagent for CDK1 or a kit thereof.

5. A kit for treating advanced gastrointestinal stromal tumors, comprising:

(a1) A first container, and an inhibitor of a CDK1 gene or protein thereof, or a medicament comprising an inhibitor of a CDK1 gene or protein thereof, in the first container, said inhibitor being RO-3306;

(b1) A second container, and a tyrosine kinase inhibitor, or a medicament containing a tyrosine kinase inhibitor, in the second container, wherein the tyrosine kinase inhibitor is imatinib.

6. The kit of claim 5, further comprising (d 1) a fourth container, and a detection reagent for CDK1 located in the fourth container.

7. Use of a pharmaceutical composition according to claim 2 or a combination of products according to claim 3 or a kit according to claim 5 for the preparation of (i) a medicament for inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) a medicament for preventing and/or treating advanced gastrointestinal stromal tumors.

8. A method of non-therapeutically inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells in vitro comprising the steps of: culturing advanced gastrointestinal stromal tumor cells in the presence of a CDK1 gene or protein inhibitor thereof, wherein the inhibitor is RO-3306, thereby inhibiting the growth or proliferation of gastrointestinal stromal tumor cells.

9. The method of claim 8, wherein the advanced gastrointestinal stromal cells highly express CDK1 protein.

10. The method of claim 8, wherein the advanced gastrointestinal stromal cells are cultured cells in vitro.

11. A method of screening for candidate compounds for the prevention and/or treatment of advanced gastrointestinal stromal tumors, the method comprising the steps of:

(a) In a test group, adding a test compound into a culture system of cells, and observing the expression level E1 and/or activity A1 of CDK1 in the cells of the test group; in the control group, no test compound was added to the culture system of the same cells, and the expression level E0 and/or activity A0 of CDK1 in the cells of the control group was observed;

Wherein, if the expression level E1 and/or activity A1 of CDK1 in the cells in the test group is significantly lower than that in the control group, the test compound is a candidate compound for preventing and/or treating advanced gastrointestinal stromal tumor with an inhibitory effect on the expression and/or activity of CDK1, and the cells are advanced gastrointestinal stromal tumor cells, and the candidate compound is RO-3306.

12. The method of claim 11, wherein the cells are cultured in vitro.