WO2020143424A1

WO2020143424A1 - Gastrointestinal stromal tumor target depdc5 and application thereof in diagnosis and treatment

Info

Publication number: WO2020143424A1
Application number: PCT/CN2019/126730
Authority: WO
Inventors: 王跃祥; 庞裕智
Original assignee: 中国科学院上海营养与健康研究所
Priority date: 2019-01-08
Filing date: 2019-12-19
Publication date: 2020-07-16
Also published as: CN111187834A; CN111187834B

Abstract

The present invention provides a gastrointestinal stromal tumor target DEPDC5 and an application thereof in diagnosis and treatment. Specifically, provided is a use of a gene, mRNA, cDNA, or a protein of DEPDC5, or a detection reagent thereof for (i) serving as markers used to detect primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors and/or (ii) preparing diagnostic reagents or kits used to detect primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors. Further provided is an application of the DEPDC5 gene, a protein thereof, or an agonist thereof in the treatment of gastrointestinal stromal tumors.

Description

The target of gastrointestinal stromal tumor DEPDC5 and its application in diagnosis and treatment

Technical field

The invention relates to the field of oncology and diagnosis. More specifically, the present invention relates to DEPDC5, the target of gastrointestinal stromal tumors, and its use in diagnosis and treatment.

Background technique

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the gastrointestinal tract. Gastrointestinal stromal tumors are divided into primary and metastatic gastrointestinal stromal tumors based on the presence or absence of metastasis. Primary gastrointestinal stromal tumors are divided according to pathological indicators (tumor size, number of mitosis per high power field, anatomical position, etc.) Low-risk, medium-risk and high-risk gastrointestinal stromal tumors, such as high-risk stromal tumor refers 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. Clinically, the treatment principles and schemes of stromal tumors with different risk levels are different. After receiving the standard treatment regimen, there are still some clinically moderate or even low-risk gastrointestinal stromal tumors with poor efficacy (embodied as tumor recurrence or even metastasis). Therefore, there is an urgent need in the field to develop targets for gastrointestinal stromal tumors that have value in assessing patient risk levels.

Gastrointestinal stromal tumors are often misdiagnosed as other gastrointestinal tumors (such as gastrointestinal smooth muscle tumors, gastrointestinal schwannoma, etc.), so the field needs to develop targets with differential diagnosis of gastrointestinal stromal tumors

About 80% of gastrointestinal stromal tumors contain KIT activating mutations. Molecular targeted therapy targeting KIT oncoproteins has revolutionized the treatment of gastrointestinal stromal tumors, especially stromal tumors during progression, but gastrointestinal stromal tumors Molecular heterogeneity among individuals leads to highly inconsistent responses of different individuals to targeted therapy. Genetic testing plays an important role in predicting the therapeutic effect of gastrointestinal stromal tumor targeted therapy and disease prognosis. At present, for patients with gastrointestinal stromal tumors receiving targeted therapy, almost all patients develop drug resistance. How to improve the efficacy of targeted therapy drugs represented by imatinib is urgently needed to be solved in clinical medicine and basic medicine. The problem.

Therefore, there is an urgent need in the art to develop therapeutic targets for gastrointestinal stromal tumors.

Summary of the invention

The object of the present invention is to provide a target for gastrointestinal stromal tumors with diagnostic or combined diagnostic value and therapeutic effect.

Another object of the present invention is to provide molecular markers for the clinical differential diagnosis of gastrointestinal stromal tumors based on the inactivation mutation of DEPDC5 gene in various human tumors. (Differentiation of tumors), a diagnostic kit can be developed to diagnose gastrointestinal stromal tumors; molecular markers can be provided for the evaluation of gastrointestinal stromal tumor risk levels.

Another object of the present invention is based on the role of DEPDC5 protein and its signaling pathway in the malignant progression of gastrointestinal stromal tumors. DEPDC5 protein and its signaling pathway are targets for the treatment of gastrointestinal stromal tumors. Gastrointestinal stromal tumors provide individualized treatment.

In the first aspect of the present invention, there is provided a use of DEPDC5 gene, mRNA, cDNA, or protein or its detection reagent, (i) for detecting primary gastrointestinal stromal tumor or advanced gastrointestinal stromal Tumor markers; and/or (ii) for the preparation of diagnostic reagents or kits for detecting primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.

In another preferred example, the diagnostic reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

In another preferred example, the protein includes a full-length protein or a protein fragment.

In another preferred example, the DEPDC5 gene, mRNA, cDNA, or protein is derived from mammals, preferably from rodents (such as mice, rats), primates and humans, more preferably, From patients diagnosed with primary gastrointestinal stromal tumors or patients diagnosed with advanced gastrointestinal stromal tumors.

In another preferred example, the DEPDC5 gene, mRNA, cDNA, or protein is derived from patients with primary or advanced gastrointestinal stromal tumors.

In another preferred example, the DEPDC5 gene, mRNA, cDNA, or protein is a mutant DEPDC5 gene, mRNA, cDNA, or protein.

In another preferred example, the mutation is selected from the group consisting of frameshift mutation, deletion mutation, point mutation, nonsense mutation, missense mutation, gene rearrangement, or a combination thereof.

In another preferred example, the DEPDC5 gene contains one or more gene mutation sites selected from the group (Table A):

Table A

c.80_105del26；c.106G>Ac.80_105del26; c.106G>A
c.2347C>Tc.2347C>T
c.1_4719del4719c.1_4719del4719
c.1_3237del3237c.1_3237del3237
c.1_2356del2356c.1_2356del2356
c.1_1693del1693c.1_1693del1693

Among them, the nucleotide position number is based on the wild-type human DEPDC5 coding gene (mRNA) sequence (NM_014662).

In another preferred example, the accession number of the DEPDC5 gene is NG_034067.

In another preferred example, the accession number of the DEPDC5mRNA is NM_014662.

In another preferred example, the accession number of the DEPDC5 protein is NP_055477.

In another preferred example, the detection includes solid tumor sample detection and normal tissue (paraneoplastic tissue) sample detection.

In another preferred example, the test is a blood sample test and/or a serum sample test.

In another preferred example, the detection reagent includes a specific antibody to DEPDC5, a specific binding molecule to DEPDC5, a specific amplification primer, a probe or a chip.

In another preferred example, the detection reagent is selected from the group consisting of antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips), protein chips, or a combination thereof.

In another preferred example, the kit contains one or more reagents selected from the group consisting of:

(a) Specific primers for DEPDC5 gene;

(b) specific probes for detecting one or more mutation sites of the gene;

(c) a chip for detecting one or more mutation sites of the gene;

(d) Specific antibodies for detecting amino acid mutations corresponding to one or more mutation sites of the gene.

In another preferred example, the DEPDC5 protein or its specific antibody or specific binding molecule is conjugated or bears a detectable label.

In another preferred example, the detectable label is selected from the group consisting of chromophore, chemiluminescent group, fluorophore, isotope or enzyme.

In another preferred example, the specific antibody of DEPDC5 is a monoclonal antibody or a polyclonal antibody.

In another preferred example, the DEPDC5 protein further includes a derivative of DEPDC5 protein.

In another preferred example, the derivative of the DEPDC5 protein includes a modified DEPDC5 protein, a protein molecule having an amino acid sequence homologous to the natural DEPDC5 protein and having natural DEPDC5 protein activity, and a fusion protein containing the amino acid sequence of the DEPDC5 protein.

In another preferred example, the modified DEPDC5 protein is a PEGylated DEPDC5 protein.

In another preferred example, the “protein molecule having an amino acid sequence homologous to the natural DEPDC5 protein and having natural DEPDC5 protein activity” means that its amino acid sequence has ≥85% homology compared to the DEPDC5 protein, preferably ≥90% homology, more preferably ≥95% homology, best ≥98% homology; and protein molecules with natural DEPDC5 protein activity.

In another preferred example, the detection reagent or kit is also used to distinguish between (a) gastrointestinal stromal tumors and adjacent tissues; (b) gastrointestinal stromal tumors and other gastrointestinal tumors (such as digestion Tract smooth muscle tumors, gastrointestinal schwannoma, gastrointestinal cancer).

In another preferred example, the primary gastrointestinal stromal tumors include low-risk and medium-risk gastrointestinal stromal tumors.

In another preferred example, the advanced gastrointestinal stromal tumors include high-risk gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors.

In another preferred example, the advanced gastrointestinal stromal tumors also include primary tumors in high-risk gastrointestinal stromal tumors, metastatic tumors formed one year later, and metastatic gastrointestinal stromal tumors. Or multiple (eg, 1 and more, preferably, 2 and more, more preferably, 5 and more) different metastases.

In the second aspect of the present invention, a diagnostic kit for detecting the risk of recurrence and metastasis of primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors is provided. The kit includes a container, so The container contains a detection reagent for detecting DEPDC5 gene, mRNA, cDNA, or protein; and a label or instructions indicating that the kit is used to detect primary gastrointestinal stromal tumor or advanced stomach Intestinal stromal tumors.

Table A

In another preferred example, the DEPDC5 has one or more amino acid residue mutations selected from the group consisting of:

p.P27fs;

p.Q783*;

The corresponding promoter and translation start site of the DEPDC5 protein are deleted;

Among them, the amino acid position number is based on the wild-type human DEPDC5 protein sequence (NP_055477).

In another preferred example, the detection of advanced gastrointestinal stromal tumor refers to judging the possibility of recurrence and metastasis of advanced gastrointestinal stromal tumor.

In another preferred example, the judgment includes pre-judgment (prediction).

In another preferred example, the detection reagent for detecting DEPDC5 gene, mRNA, cDNA, or protein includes:

(a). Specific antibodies against DEPDC5 protein; and/or

(b). Specific primers that specifically amplify the mRNA or cDNA of DEPDC5.

In another preferred example, the detection reagent includes one or more reagents selected from the group consisting of:

(a) Specific primers for DEPDC5 gene;

(b) specific probes for detecting one or more mutation sites of the gene;

(c) a chip for detecting one or more mutation sites of the gene;

In another preferred example, the detection is a solid tumor tissue sample detection.

In another preferred example, the DEPDC5 gene, mRNA, cDNA, or protein or its detection reagent can be used as a control or reference.

In another preferred example, the label or instructions indicate that the kit is used for:

(i) Detection of primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors; and/or

(ii) distinguish gastrointestinal stromal tumors from adjacent tissues; and/or

(iii) distinguish gastrointestinal stromal tumors from other gastrointestinal tumors (eg gastrointestinal smooth muscle tumors, gastrointestinal schwannomas, gastrointestinal cancers); and/or

(iv) Distinguish the risk level of gastrointestinal stromal tumor recurrence and metastasis.

In another preferred example, the detection object is a human or non-human mammal.

In another preferred example, the kit is also used to predict the survival time or prognosis of patients with gastrointestinal stromal tumors.

In a third aspect of the present invention, there is provided a method for detecting primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor, the method comprising:

a) Provide test samples from subjects;

b) Detect the expression level of DEPDC5 protein in the test sample and/or the mutation status of DEPDC5 protein; and

c) Compare the expression level of DEPDC5 protein measured in step b) with the control,

Compared with the control, the expression level of DEPDC5 protein in the sample is lower than the reference value, indicating that the probability of the subject suffering from gastrointestinal stromal tumor or metastatic gastrointestinal stromal tumor is higher than that of the general population ( Control group); or

If the DEPDC5 protein contains one or more amino acid mutation sites selected from the following group, it indicates that the subject has a higher risk of primary gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors than the general population ( Control group);

p.P27fs;

p.Q783*;

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

In another preferred example, the test sample is a primary gastrointestinal stromal tumor or a solid tumor tissue sample of advanced gastrointestinal stromal tumor.

In another preferred example, the test sample includes a blood sample and/or a serum sample of primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor.

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

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

In another preferred example, the reference value is the relative expression level of DEPDC5 in the sample.

In another preferred example, the reference value is 0.4.

In another preferred example, the expression level of DEPDC5 protein in the sample is detected by RT-PCR or immunohistochemistry.

In a fourth aspect of the present invention, a method for determining a treatment plan is provided, including:

a) Provide test samples from subjects;

c) Determine the treatment plan based on the expression level of DEPDC5 protein in the sample and/or the mutation status of DEPDC5 protein.

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

In another preferred example, when the expression level of DEPDC5 protein in the sample is lower than the reference value, it indicates that the subject has a higher probability of having primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor The general population (control group), the treatment regimen includes DEPDC5 gene therapy, DEPDC5 agonist therapy, and/or targeted downstream molecular mechanism therapy.

In another preferred example, when the DEPDC5 protein contains one or more amino acid mutation sites selected from the group below, it indicates that the subject has primary gastrointestinal stromal tumor or metastatic gastrointestinal stromal tumor The odds are higher than the general population (control group), the treatment regimen includes DEPDC5 gene therapy, DEPDC5 agonist therapy, and/or targeted downstream molecular mechanism therapy.

In another preferred example, the DEPDC5 gene therapy, DEPDC5 agonist therapy and/or targeted downstream molecular mechanism therapy are selected from the group consisting of:

DEPDC5 gene therapy: human DEPDC5 gene or its protein;

DEPDC5 agonist: a carrier expressing DEPDC5 protein, a small molecule compound, or a combination thereof;

Targeted downstream molecular mechanism therapy: mTORC1 inhibitor.

In another preferred example, the mTORC1 inhibitor is selected from the group consisting of Everolimus, Rapamycin, Temsirolimus, AZD8055, WYE-354, WYE- 125132, WYE-687, WAY-600, XL388, CZ415, Zotarolimus, Tacrolimus, Palomid 529, Ridaforolimus (Deforolimus or MK-8669), Dactolisib (BEZ235 or NVP-BEZ235), or a combination thereof.

In another preferred example, when the subject has a higher incidence of primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors than the general population (control group), the treatment regimen also includes DEPDC5 gene therapy, agonist therapy and/or targeted downstream molecular mechanism therapy; in combination with other drugs for the treatment of gastrointestinal stromal tumors.

In another preferred example, the other drugs for the treatment of gastrointestinal stromal tumors are selected from the group consisting of imatinib, sunitinib, rifafenib, Avapritinib (BLU-285), Ripretinib (DCC-2618 ), or a combination thereof.

In another preferred example, the treatment regimen also includes other treatments for advanced gastrointestinal stromal tumors.

In another preferred example, the treatment of other advanced gastrointestinal stromal tumors is selected from the group consisting of imatinib, sunitinib, rifafenib, Avapritinib (BLU-285), Ripretinib (DCC -2618), or a combination thereof.

In the fifth aspect of the present invention, there is provided the use of the DEPDC5 gene, or its protein, or its agonist, for the preparation and prevention of primary gastrointestinal stromal tumor or advanced gastrointestinal stromal Tumor drugs.

In another preferred example, the activator is a substance that promotes the expression of the gene or increases the activity of the gene expression product (protein).

In another preferred example, the agonist is selected from the group consisting of DEPDC5 protein, a nucleic acid expressing DEPDC5 protein, a vector expressing DEPDC5 protein, a small molecule compound, or a combination thereof.

In the sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) DEPDC5 gene, or its protein, or its agonist;

(b) Other drugs to prevent and/or treat primary gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors; and

(c) A pharmaceutically acceptable carrier.

In another preferred example, the other drugs for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors are selected from the group consisting of imatinib, sunitinib, Regefenib, Avapritinib (BLU-285), Ripretinib (DCC-2618), or a combination thereof.

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

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

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

In another preferred example, in the product combination, the component (a) and the component (b) account for 0.01-99.99% by weight of the total weight of the product combination, preferably 0.1-90% by weight, more preferably Ground 1-80wt%.

In another preferred example, the dosage form of the pharmaceutical composition includes an injection dosage form and an oral dosage form.

In another preferred example, the oral dosage form includes tablets, capsules, films, and granules.

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

In a seventh aspect of the present invention, a kit is provided, including:

(a1) The first container, and the DEPDC5 gene, or its protein, or its agonist, or a drug containing the DEPDC5 gene, or its protein, or its agonist, located in the first container;

(b1) a second container, and other medicines for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors located in the second container, or containing other preventive and/or Drugs for treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.

In another preferred example, the first container and the second container are the same or different containers.

In another preferred example, the medicine in the first container is a unilateral preparation containing the DEPDC5 gene, or its protein, or its agonist.

In another preferred example, the medicine in the second container is a unilateral preparation containing other medicines for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.

In another preferred example, the pharmaceutical dosage form is an oral dosage form or an injection dosage form.

In another preferred example, the kit also contains instructions.

In another preferred example, the description records one or more descriptions selected from the following group:

(a) Combining the DEPDC5 gene, or its protein, or its agonist, and other drugs to prevent and/or treat primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors Methods for primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors;

(b) Combine the DEPDC5 gene, or its protein, or its agonist, and other drugs for the prevention and/or treatment of primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors to simultaneously detect the primary stomach Intestinal stromal tumor or advanced gastrointestinal stromal tumor patients with DEPDC5 protein expression level or DEPDC5 protein mutation status to treat primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor .

In the eighth aspect of the present invention, there is provided the use of the pharmaceutical composition according to the sixth aspect of the present invention or the kit according to the seventh aspect of the present invention, for the prevention and/or treatment of primary gastrointestinal tract Gliomas or advanced gastrointestinal stromal tumors.

In another preferred example, in the pharmaceutical composition, the concentration of the DEPDC5 gene, its protein, or its agonist is 40-400000ng/ml, preferably 400-40000ng/ml, more preferably, 2000 -8000ng/ml.

In another preferred example, in the pharmaceutical composition, the effective concentration of the other drugs for preventing and/or treating primary gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors is 10-100000 ng/ ml, preferably, 100-10000ng/ml, more preferably, 500-2000ng/ml.

In another preferred example, the pharmaceutical composition or kit includes (a) DEPDC5 gene, or its protein, or its agonist; and other prevention and/or treatment of primary gastrointestinal stromal tumor or advanced stage Drugs for gastrointestinal stromal tumors; and (b) pharmaceutically acceptable carriers.

In another preferred example, in the pharmaceutical composition or kit, the DEPDC5 gene, or its protein, or its agonist; and other prevention and/or treatment of primary gastrointestinal stromal tumor or advanced stage The gastrointestinal stromal tumor drug accounts for 0.01-99.99% by weight of the total weight of the pharmaceutical composition or kit, preferably 0.1-90% by weight, more preferably 1-80% by weight.

In a ninth aspect of the present invention, a method for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors is provided, including:

The DEPDC5 gene, its protein, or its agonist; or the pharmaceutical composition according to the sixth aspect of the present invention or the kit according to the seventh aspect of the present invention is administered to a subject in need.

In another preferred example, the subject includes a human or non-human mammal with primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor.

In another preferred example, the non-human mammal includes rodents and primates, preferably mice, rats, rabbits, and monkeys.

In another preferred example, the administration dose of the DEPDC5 gene, or its protein, or its agonist is 0.24-2400 mg/kg body weight, preferably 2.4-240 mg/kg body weight, and most preferably 12-48 mg/ kg body weight.

In another preferred example, the dose of the other drugs for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors is 0.06-600 mg/kg body weight, preferably 0.6-60 mg/kg body weight, optimally 3-12 mg/kg body weight.

In another preferred example, the application frequency of the DEPDC5 gene, or its protein, or its agonist is 1-4 times/day, preferably 1-2 times/day.

In another preferred example, the administration frequency of the other drugs for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors is 1-4 times/day, preferably 1-2 times/day.

In another preferred example, the interval between two consecutive administrations of the DEPDC5 gene, or its protein, or its agonist is 6 hours or more, preferably 12 hours or more, and most preferably 24 hours or more.

In another preferred example, the interval between two consecutive administrations of the other drugs for preventing and/or treating gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors is more than 6 hours, preferably 12 hours or more, preferably 24 hours or more.

In another preferred example, the DEPDC5 gene, or its protein, or its agonist and other drugs for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors are simultaneous or sequential Apply.

In a tenth aspect of the present invention, a method for inhibiting the growth or proliferation of gastrointestinal stromal tumors in vitro is provided, comprising the steps of: cultivating the gastrointestinal tract in the presence of the DEPDC5 gene, or its protein, or its agonist Stromal tumor cells, thereby inhibiting the growth or proliferation of gastrointestinal stromal tumor cells.

In another preferred example, the gastrointestinal stromal tumor cells include primary gastrointestinal stromal tumor cells and/or advanced gastrointestinal stromal tumor cells.

In another preferred example, the method further includes adding other preventive and/or treatments to the primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor to the culture system of gastrointestinal stromal tumor cells Drugs, thereby inhibiting the growth or proliferation of gastrointestinal stromal tumor cells.

In another preferred example, the gastrointestinal stromal tumor cells are cells cultured in vitro.

In an eleventh aspect of the present invention, there is provided a method for screening candidate compounds for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors, the method comprising the steps of:

(a) In the test group, add the test compound to the cell culture system, and observe the expression level and/or activity of DEPDC5 in the cells of the test group; in the control group, do not add the test in the same cell culture system Compound, and observe the expression level and/or activity of DEPDC5 in the cells of the control group;

Among them, if the DEPDC5 expression and/or activity of the cells in the test group is higher than that of the control group, it indicates that the test compound is a preventive and/or therapeutic treatment for primary gastrointestinal tract that promotes the expression and/or activity of DEPDC5 Candidate compounds for stromal tumors or advanced gastrointestinal stromal tumors.

In another preferred example, the expression level of DEPDC5 is obtained by RT-PCR or immunohistochemical detection.

In another preferred example, the method further includes the steps of:

(b) For the candidate compound obtained in step (a), further test its inhibitory effect on cell growth or proliferation of primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor; and/or further test Does it have an up-regulation effect on the DEPDC5 gene?

In another preferred example, step (b) includes the step of: in the test group, the test compound is added to the culture system of cells of primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor, and observation The number and/or growth of cells in primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor; in the control group, in the primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor No test compound is added to the culture system of tumor cells, and the number and/or growth of cells in primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors are observed; where, if the test group is primary Gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors, the number or growth rate of cells is less than the control group, indicating that the test compound is a primary gastrointestinal stromal tumor or advanced gastrointestinal tract Candidate compounds for the prevention and/or treatment of primary gastrointestinal stromal tumors or cells of advanced gastrointestinal stromal tumors with an inhibitory effect on the growth or proliferation of the cells of gliomas.

It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (eg, embodiments) can be combined with each other, thereby forming a new or preferred technical solution. Due to space limitations, I will not repeat them here.

BRIEF DESCRIPTION

Figure 1 shows (a) 7 out of 40 GIST patients (17.5%) were found to have DEPDC5 inactivating mutations. Arrows indicate single nucleotide or insertion deletion mutations, and blue horizontal lines indicate deleted exon regions. (b) The DEPDC5 gene mutation only occurs in the patient's tumor tissue, but does not exist in normal tissue, proving that the DEPDC5 gene mutation is a somatic mutation. (c) Sequencing results showed that frameshift mutation of DEPDC5 in GIST of patient 29. (d) SNP whole-genome microarray analysis showed that there was a homozygous deletion of the DEPDC5 gene in patients with GIST, but no normal tissue. (e) qPCR results showed that the corresponding exon region of DEPDC5 in GIST was deleted compared to normal tissues. (f) FISH analysis showed that there was a homozygous deletion of the DEPDC5 gene in GIST cells, but not in normal cells.

Figure 2 shows (a) DEPDC5 gene deletion mutations exist in both the primary lesion and metastatic lesion tissue formed in one GIST patient, but normal tissue does not. (b-c) DEPDC5 gene frameshift mutations existed in each metastasis of other patients with GIST and in various tissues of metastasis, but not in normal tissues. (d) After continuous in vivo xenotransplantation of GIST882 or continuous cultivation in vitro with different drugs for a period of time, the DEPDC5 gene deletion mutation can still be detected. (e) The relative expression of DEPDC5 gene decreases as the risk of GIST patients increases.

Figure 3 shows that the mutation frequency of DEPDC5 gene in GISTs is significantly higher than other sarcomas.

Figure 4 shows that compared with the control group, the growth rate of GIST882 cells after DEPDC5 introduction was significantly slower in vitro (a), cell viability was significantly reduced (b), intracellular PCNA expression was reduced (c), and the cell cycle G0/G1 phase ratio Increased, while the proportion of S phase decreased (d), suggesting that the cell proliferation capacity was reduced, tumor growth rate (e) and tumor size (f) were inhibited in nude mice, and cell division activity was significantly reduced (g).

Figure 5 shows that compared with the control group, GIST430 knocked out DEPDC5 in vitro after cell growth rate increased (a), intracellular PCNA expression increased (b), cell cycle G0/G1 phase ratio decreased (c), suggesting DEPDC5 knock Promotes cell proliferation after removal.

Figure 6 shows (a) GSEA analysis shows that compared with the control group, the mTORC1 signaling pathway after the introduction of DEPDC5 and the cell cycle (E2F target gene, G2M cell cycle checkpoint, mitotic spindle) related genes have a significant decrease in the transcription level expression. Enrichment. (b) Western blotting showed that compared with the control group, the phosphorylation level of mTOR downstream protein p70S6K and S6 decreased and the phosphorylation level of AKT increased after DEPDC5 was introduced into GIST882 cells, indicating that the mTORC1 signaling pathway was inhibited, while KIT or MAPK protein phosphorylation The level has not changed significantly. (c) Western blotting showed that the phosphorylation level of mTOR downstream protein p70S6K and S6 increased after knocking out DEPDC5 in GIST-T1 or GIST430 cells compared with the control group, reflecting the activation of the mTORC1 signaling pathway and the phosphorylation level of KIT or MAPK protein No obvious changes.

Figure 7 shows (a) the dose-response curve showing that, compared with the control group, the half-inhibitory concentration (IC ₅₀ ) of Imatinib was significantly reduced after the introduction of DEPDC5, demonstrating that DEPDC5-introduced GIST882 cells were more sensitive to Imatinib. (b) Immunoblotting shows that Imatinib and DEPDC5 act on KIT and mTOR signaling pathways respectively. Imatinib can inhibit the downstream proteins of mTOR by inhibiting KIT. (c) The simultaneous effect of the introduction of DEPDC5 and the treatment of Imatinib on GIST882 cells is greater than the separate effects of the two, indicating that DEPDC5 protein and Imatinib have a synergistic effect. (de) The combined effect of Everolimus and Imatinib on GIST882 cells is greater than that of the single drug, indicating that there is a synergistic effect between the two; when DEPDC5 is introduced, the synergistic effect of the two drugs is reduced. (f) show dose-response curve, compared with the control group, DEPDC5 Imatinib increased after knockout of the IC _50, GIST430 cells showed DEPDC5 knockout more resistant to Imatinib. (g) The combined effect of Everolimus and low concentration of Imatinib has a synergistic effect on the inhibition of GIST430 cells; when DEPDC5 is knocked out, the synergistic effect of the two drugs is enhanced.

detailed description

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that there is a DEPDC5 gene mutation in primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors, but no DEPDC5 gene in normal tissues mutation. Therefore, the DEPDC5 gene can be used as a marker for detecting primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors. Moreover, the applicant has also unexpectedly found that the DEPDC5 gene or its protein, or its agonist, can increase the sensitivity of gastrointestinal stromal tumors to drugs for the treatment of gastrointestinal stromal tumors, thereby improving the gastrointestinal stromal tumors. Therapeutic effect, and the combination of DEPDC5 gene or its protein, or its agonist and gastrointestinal stromal tumors has a significant synergistic effect. In addition, the DEPDC5 gene or its protein alone or its agonist can also be effective in the treatment of gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors. On this basis, the present inventor has completed the present invention.

As used herein, the structural formula of the term "AZD8055" is

As used herein, the structural formula of the term "WYE-354" is

As used herein, the structural formula of the term "WYE-125132" is

As used herein, the structural formula of the term "WYE-687" is

As used herein, the structural formula of the term "WAY-600" is

As used herein, the structural formula of the term "XL388" is

As used herein, the structural formula of the term "CZ415" is

As used herein, the structural formula of the term "Zotarolimus" is

As used herein, the structural formula of the term "Tacrolimus" is

As used herein, the structural formula of the term "Palomid 529" is

As used herein, the structural formula of the term "Ridaforolimus (Deforolimus or MK-8669)" is

As used herein, the structural formula of the term "Dactolisib (BEZ235 or NVP-BEZ235)" is

As used herein, the term "Avapritinib (BLU-285)" structural formula is

As used herein, the structural formula of the term "Ripretinib (DCC-2618)" is

Gastrointestinal stromal tumor

Gastrointestinal stromal tumors are the most common mesenchymal tumors in the gastrointestinal tract. Clinically symptomatic gastrointestinal stromal tumors occur in adults over 45 years old. Gastrointestinal stromal tumors can occur throughout The digestive tract, but most of them occur in the stomach and small intestine, and clinical stromal tumors are the most common sarcoma, with an annual incidence of 10-20 per 1 million people. With the progress of endoscopic technology and imaging technology, more and more small gastrointestinal stromal tumors are found. A number of pathological studies have confirmed that tiny gastrointestinal stromal tumors less than 1cm in diameter are common among middle-aged and elderly people, and the discovery rate can reach 35%. With the aging of the world population increasing, it is estimated that China’s tiny gastrointestinal stromal tumors There are 100 million patients with tumors.

The main pathogenesis of gastrointestinal stromal tumors is the activation mutation of the proto-oncogene KIT in Kahal interstitial cells, which abnormally activates downstream signaling pathways, including the MAPK pathway and PI3K-AKT pathway, which makes the cell survival, growth and proliferation out of control .

Advanced gastrointestinal stromal tumor

Gastrointestinal stromal tumors are divided into primary gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors according to the presence or absence of metastasis. Primary gastrointestinal stromal tumors are based on pathological indicators (tumor size, number of mitosis per high power field of view) , Anatomical location, etc.) is divided into low-risk, medium-risk and high-risk gastrointestinal stromal tumors, such as high-risk gastrointestinal stromal tumor refers 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 advanced gastrointestinal stromal tumors is poor, and clinical treatment is urgently needed. Approximately 80% of gastrointestinal stromal tumors contain KIT activating mutations. Molecular targeted therapy targeting KIT oncoproteins has revolutionized the treatment of gliomas during progression, but molecular heterogeneity among individuals with gastrointestinal stromal tumors has led to Different individuals respond highly inconsistently to targeted therapy. Genetic testing plays an important role in predicting the therapeutic effect of gastrointestinal stromal tumor targeted therapy and disease prognosis. At present, for patients with gastrointestinal stromal tumors receiving targeted therapy, almost all patients develop drug resistance. How to improve the efficacy of targeted therapy drugs represented by imatinib is urgently needed to be solved in clinical medicine and basic medicine. The problem.

sample

The term "sample" or "sample" as used herein refers to material that is specifically associated with a subject, from which specific information related to the subject can be determined, calculated, or inferred. The sample may be wholly or partially composed of biological material from the subject. The sample may also be a material that has been in contact with the subject in a manner that allows the test to be performed on the sample to provide information about the subject. The sample may also be a material that has been in contact with other materials, which is not the subject's, but enables the first material to be subsequently tested to determine information about the subject, for example the sample may be a probe or an anatomy Knife cleaning fluid. The sample may be a source of biological material that is not in contact with the subject, as long as a person skilled in the art can still determine the information related to the subject from the sample.

expression

As used herein, the term "expression" includes the production of mRNA from genes or gene parts, and includes the production of proteins encoded by RNA or genes or gene parts, and also includes the appearance of detection substances related to expression. For example, cDNA, binding ligands (such as antibodies) to genes or other oligonucleotides, proteins or protein fragments, and the coloring portion of the binding ligand are all included in the scope of the term "expression". Therefore, the increase in the density of the upper half dots in immunoblotting, such as western blotting, is also within the scope of the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically related to a particular result when compared to the analysis result. In a preferred embodiment, the reference value is determined based on statistical analysis of studies comparing the expression of DEPDC5 protein with known clinical results. Some such studies are shown in the Examples section of this article. However, research from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. The reference value can 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 DEPDC5 in solid tumors, preferably 0.4.

DEPDC5 protein and polynucleotide

In the present invention, the terms "protein of the present invention", "DEPDC5 protein", and "DEPDC5 polypeptide" are used interchangeably, and all refer to proteins or polypeptides having the amino acid sequence of DEPDC5. They include DEPDC5 protein with or without starting methionine. In addition, the term also includes full-length DEPDC5 and fragments thereof. The DEPDC5 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.

The DEPDC5 (DEP domain-containing) 5 protein contains a DEP (Dishevelled, Egl-10, Pleckstrin) domain and a DUF3608 domain, which are commonly expressed in human tissues. The gene encoding DEPDC5 is located on the long arm of human chromosome 22.

The human DEPDC5 protein has a total length of 1572 amino acids (accession number NP_055477). The full length of the mouse DEPDC5 protein is 1591 amino acids (accession number NP_001164038).

In the present invention, the terms "DEPDC5 gene" and "DEPDC5 polynucleotide" are used interchangeably, and all refer to a nucleic acid sequence having a DEPDC5 nucleotide sequence.

The human DEPDC5 gene has a total genome length of 16,084 bp (NCBI GenBank accession number NG_034067; Gene ID: 9681), and its transcript mRNA sequence is 5,326 bp (NCBI GenBank accession number NM_014662).

The full-length genome of the mouse DEPDC5 gene is 130536 bp (NCBI GenBank accession number is Gene ID: 277854), and its transcribed product mRNA sequence is 7944 bp (NCBI GenBank accession number is NM_001170567).

The similarity between human and murine DEPDC5 at the DNA level is 94.9%, and the protein sequence similarity is 94.2%.

It should be understood that when encoding the same amino acid, substitution of nucleotides in codons is acceptable. It should also be understood that when conservative amino acid substitutions are generated by nucleotide substitutions, nucleotide substitutions are also acceptable.

When the amino acid fragment of DEPDC5 is obtained, a nucleic acid sequence encoding it can be constructed from it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the DEPDC5 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or cDNA prepared according to conventional methods known to those skilled in the art The library serves as a template and the relevant sequences are amplified. When the sequence is long, 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 sequence is obtained, the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, synthetic methods can also be used to synthesize the relevant sequences, especially when the length of the fragments is short. Generally, a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.

At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

Through conventional recombinant DNA technology, the polynucleotide sequences of the present invention can be used to express or produce recombinant DEPDC5 polypeptides. Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) of the present invention encoding human DEPDC5 polypeptide, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify proteins from culture medium or cells.

In the present invention, the DEPDC5 polynucleotide sequence can be inserted into a recombinant expression vector. In short, as long as it can replicate and stabilize in the host, any plasmid and vector can be used. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing DEPDC5 encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology and so on. The DNA sequence can be effectively linked to an appropriate promoter in an expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

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

Vectors containing the appropriate DNA sequences and appropriate promoters or control sequences described above can be used to transform appropriate host cells so that they can express proteins.

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

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 prokaryotic organism such as E. coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The procedures used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by a conventional method and express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The cultivation is carried out under conditions suitable for the growth of host cells. When the host cell grows to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.

The recombinant polypeptide in the above method may be expressed in a cell, on a cell membrane, or secreted out of the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Gene mutation

Gene mutation (DNA sequence mutation, gene mutation) is due to the addition, deletion or replacement of base pairs in the DNA molecule, resulting in changes in the structure of the gene.

The site where the gene mutation occurs is the mutation site in this article, and base addition, deletion or replacement can occur at the mutation site.

For example, "chr11:g.67051695A>C" means that the position of g.67051695 on human chromosome 11 is mutated from A to C.

"Chr11:g.64577368_64577374(GCGGGTC)>-" means the deletion of GCGGGTC at positions g.64577368 to 64577374 on human chromosome 11.

"Chr19:g.14938120->T" means that the base T is added at the position of g.14938120 on human chromosome 19.

In the present invention, c.80_105del26; c.106G>A refers to the deletion of the 80th to 105th positions of the gene (mRNA) encoding DEPDC5, and the guanine at position 106 is mutated to adenine.

c. 2347C>T refers to the mutation of cytosine at position 2347 of the gene (mRNA) encoding DEPDC5 to thymine.

c. 1_4719del4719 refers to the deletion of positions 1 to 4719 of the gene (mRNA) encoding DEPDC5.

c. 1_3237del3237 refers to the deletion of positions 1 to 3237 of the gene (mRNA) encoding DEPDC5.

c. 1_2356del2356 refers to the deletion of positions 1 to 2356 of the gene (mRNA) encoding DEPDC5.

c. 1_1693del1693 refers to the deletion of positions 1 to 1693 of the gene (mRNA) encoding DEPDC5.

A typical type of genetic mutation is SNV, which is a single nucleotide mutation, especially SNV that causes amino acid mutations.

In addition, in the present invention, there are cases where amino acid mutation or DEPDC5 protein is not expressed (promoter and translation start site are deleted).

For example, "p.P27fs" means that the DEPDC5 protein starts a frameshift mutation from the 27th position of proline and forms a stop code after the 11th amino acid downstream.

"P.Q783*" means that the glutamine at position 783 of the DEPDC5 protein is mutated to a stop code.

Single Nucleotide Variation (SNV)

Single nucleotide variation is a variation of the DNA sequence in the human genome and has gained increasing importance in various biological and biomedical applications. SNVs can be used to explore the evolutionary history of human populations and analyze forensic samples, so they play an important role in genetics. Pharmacogenetics uses these DNA variations to elucidate the underlying genetic factors that make up the efficacy or adverse events of different drugs.

The present invention relates to the identification of single nucleotide variants (SNVs) of specific diseases that are specifically identified as being associated with pituitary adenoma, and therefore, or before the presence of disease symptoms, these individuals can be intervened, such as dietary changes, exercise and/or medical treatement. Identification of SNVs involving pituitary adenoma helps to better understand the disease process and improve diagnostic and therapeutic reagents.

As used herein, the term "SNV" refers to a single nucleotide variation at a specific position in the human genome that differs between groups of individuals. In the present invention, the SNV can be determined by its name or by being located in a specific sequence. For example, SNV "[G/A]" means that the nucleotide base (or allele) at this position of the sequence may be guanine or adenine.

As used herein, INDEL insertion deletion marker refers to the difference in the whole genome of two parents. Compared with the other parent, one of the parents has a certain number of nucleotide insertions or deletions in the genome.

As used herein, the disclosed nucleotide sequence includes the complementary sequence of the nucleotide sequence. In addition, the term "SNV" includes any allele in a group of alleles.

As used herein, the term "allele" refers to a specific nucleotide in the nucleotide selection that defines SNV.

As used herein, the term "risk allele" refers to an allele associated with gastrointestinal stromal tumor or metastatic gastrointestinal stromal tumor disease.

As used herein, the term "haplotype" refers to a combination of specific alleles from two or more SNVs.

As used herein, the term "risk state haplotype" refers to a haplotype associated with gastrointestinal stromal tumor or metastatic gastrointestinal stromal tumor disease.

See Table A for the specific genes and the mutation sites they contain.

Table A

Unless otherwise specified, the locus numbering in the present invention is based on the human genome sequence (UCSC) hg19 version.

Polynucleotides containing mutation sites

The invention also provides a polynucleotide containing the mutation site of the invention. In a preferred embodiment of the present invention, the present invention also provides a vector and a host cell containing the polynucleotide.

As used herein, the term "polynucleotide" refers to a polymorphism of nucleotides of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Polynucleotides can have any three-dimensional structure, including single-stranded, double-stranded, and triple-helical molecular structures, and can perform any known or unknown function. As in the following non-limiting examples: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, siRNA, ribozymes, cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, Any sequence of isolated RNA, nucleic acid probes, primers. Polynucleotides can also include modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.

In another preferred example, the polynucleotide itself may further include a detection reagent for detecting the polynucleotide, including primers, probes, amplification products, or plasmids.

As used herein, the term "substantially isolated" or "isolated" polynucleotide refers to a polynucleotide that is substantially free of natural related sequences. Basically does not mean that at least 50%, preferably at least 70%, more preferably at least 80% or most preferably at least 90% are free of other naturally related substances. "Isolated polynucleotide" also includes recombinant polynucleotides.

As used herein, the term "hybridize under stringent conditions" is intended to describe hybridization conditions under which at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98 of each other Nucleotide sequences that are% identical typically remain hybridized to each other. These stringent conditions are known to those skilled in the art and can be found in Current Protocols Molecular Biology, John Wiley & Sons, N.Y (1989). A non-limiting example of stringent hybridization is hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washings at 50-65°C in 0.2xSSC, 0.1% SDS.

As used herein, the term "primer" refers to a generic term for oligonucleotides that can be paired with a template and can be used as a starting point to synthesize a DNA strand complementary to the template under the action of a DNA polymerase. The primer may be natural RNA, DNA, or any form of natural nucleotide. Primers can even be unnatural nucleotides such as LNA or ZNA. The primer is "substantially" (or "substantially") complementary to a special sequence on a strand of the template. The primer must be sufficiently complementary to a strand on the template to begin extension, but the sequence of the primer need not be completely complementary to the sequence of the template. For example, by adding a sequence that is not complementary to the template at the 5'end of a primer that is complementary to the template at the 3'end, such a primer is still substantially complementary to the template. As long as there are sufficiently long primers that can fully bind to the template, primers that are not completely complementary can also form a primer-template complex with the template to perform amplification.

As used herein, the term "vector" refers to a DNA molecule that can carry inserted DNA and can be maintained in a host cell. The vector may also be a cloning vector, a cloning vehicle, or the like. The term "vector" includes a vector whose main function is to insert a nucleic acid molecule into a cell, a replication vector whose main function is to replicate nucleic acids, and an expression vector used to transcribe and/or translate DNA or RNA, and also to provide more than one of the above functions Carrier.

As used herein, the term "host cell" refers to a single cell or cell culture, which may or has been a recipient for the integration of a vector or nucleic acid molecule and/or protein. Host cells include progeny of a single host cell, and due to natural, random, or intentional mutations, the progeny may not necessarily be exactly the same as the parent (morphologically or in total DNA complementary sequence). Host cells include cells transfected with the polynucleotide of the present invention. "Isolated host cell" refers to a host cell that has been physically separated from the organism from which it originated.

Specific antibody

In the present invention, the terms "antibody of the present invention" and "specific antibody against DEPDC5" are used interchangeably.

The invention also includes polyclonal antibodies and monoclonal antibodies specific for human DEPDC5 polypeptides, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to the human DEPDC5 gene product or fragment. Preferably, it refers to those antibodies that can bind to the human DEPDC5 gene product or fragment but do not recognize and bind to other unrelated antigen molecules. Antibodies in the present invention include those molecules that can bind to and inhibit human DEPDC5 protein, as well as those that do not affect the function of human DEPDC5 protein. The invention also includes those antibodies that bind to the human DEPDC5 gene product in modified or unmodified form.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of murine antibodies but still retain antibody portions from humans.

The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the purified human DEPDC5 gene product or its antigenic fragments can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human DEPDC5 protein or its antigenic fragments can be used to immunize animals to produce antibodies. The antibody of the present invention may be a monoclonal antibody. 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., In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies that can block the function of human DEPDC5 protein and antibodies that do not affect the function of human DEPDC5 protein. The various antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of the human DEPDC5 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to the unmodified form of the human DEPDC5 gene product can be produced by immunizing animals with the gene products produced in prokaryotic cells (such as E. Coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated) Proteins or polypeptides) can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

Anti-human DEPDC5 protein antibodies can be used in immunohistochemical techniques to detect human DEPDC5 protein in specimens (especially tissue samples or serum samples). Since the DEPDC5 protein exists in the extracellular domain, these soluble DEPDC5 extracellular domains can be the targets of serum detection when the extracellular domain sheds and enters the blood.

Detection method

The use of mutant DEPDC5 exists in solid tumor tissues and body fluids (preferably serum or blood) of gastrointestinal stromal tumors, and is closely related to the risk of recurrence and metastasis of gastrointestinal stromal tumors. The present invention also provides A method for detecting advanced gastrointestinal stromal tumors.

In a preferred example of the present invention, the present invention provides a high-throughput next-generation sequencing method for detecting mutant DEPDC5 and Sanger sequencing, fluorescence quantitative PCR (qPCR), single nucleotide polymorphism (SNP) genome analysis, In situ immunofluorescence (FISH).

Detection kit

Mutation-based DEPDC5 is associated with the recurrence and metastasis of gastrointestinal stromal tumors, that is, mutant DEPDC5 is present in gastrointestinal stromal tumor solid tumor tissues and body fluids (preferably blood or serum), so mutant DEPDC5 can be used as progress A diagnostic marker of gastrointestinal stromal tumors.

The present invention also provides a diagnostic kit for detecting the risk of primary gastrointestinal stromal tumor or gastrointestinal stromal tumor recurrence and metastasis, which contains a detection reagent for detecting DEPDC5 gene, mRNA, cDNA, or protein; and a label Or instructions, the label or instructions indicate that the kit is used to detect primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors;

Among them, the label or instruction manual states the following:

(i) Detection of gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors; and/or

(ii) distinguish gastrointestinal stromal tumors from adjacent tissues; and/or

(iii) distinguish gastrointestinal stromal tumors from other gastrointestinal tumors (such as gastrointestinal smooth muscle tumors, gastrointestinal schwannomas, gastrointestinal cancers, etc.); and/or

It should be understood that, after the present invention first discloses the correlation between the mutation site of the present invention and gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors, those skilled in the art can easily design specific amplification The amplified products containing the mutation sites are then determined by sequencing or other methods to determine whether these mutation sites are present.

Generally, the length of the primer is 15-50 bp, preferably 20-30 bp. Although it is preferred that the primer and the template sequence are completely complementary, those skilled in the art know that even if there is a certain non-complementation between the primer and the template (especially the 5′ end of the primer), it can also specifically amplify (i.e., only Amplify the desired fragment). Kits containing these primers and methods of using these primers are within the scope of the present invention, as long as the amplification products amplified by the primers contain the corresponding positions of the mutation sites of the present invention.

Although the length of the amplification product is not particularly limited, the length of the amplification product is usually 100-3000 bp, preferably 150-2000 bp, and more preferably 200-1000 bp.

The main advantages of the present invention include:

(1) For the first time, the present invention found that there are inactivating mutations of DEPDC5 (such as deletion mutations, frameshift mutations, nonsense mutations, missense mutations, etc.) in gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors, It does not exist in normal tissues.

(2) The present invention found for the first time that DEPDC5 (especially mutant DEPDC5) can be used as a marker for detecting primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.

(3) The present invention found for the first time that the relative expression of the DEPDC5 gene decreases as the risk of patients with primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors increases.

(4) The present invention found for the first time that inactivating mutations of DEPDC5 can promote the growth of primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors, promote primary gastrointestinal stromal tumors or advanced stomach Intestinal stromal tumor cell growth and activation of mTORC1 signaling pathway promote cell growth.

(5) The present invention finds for the first time that the DEPDC5 gene or its protein or its agonist can effectively treat primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.

(6) The present invention found for the first time that the DEPDC5 gene or its protein, or its agonist, can increase the sensitivity of gastrointestinal stromal tumors to drugs for the treatment of gastrointestinal stromal tumors, thereby improving the treatment of gastrointestinal stromal tumors The effect, and DEPDC5 gene or its protein, or its agonist, and gastrointestinal stromal tumor treatment drugs have a significant synergistic effect.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples generally follow conventional conditions, such as Sambrook et al. Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.

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

General method

1. Lentivirus packaging

Polyethylenime (PEI) was used to mediate 293T cell transfection. The cells were divided into 10 cm dishes at an appropriate density one day before transfection. When the cells grew to about 70-90%, the medium was replaced with serum-free medium, and the plasmid was transfected with PEI 2 hours later. The lentiviral packaging plasmids are 9μgδ8.9 and 3.5μg vsv-g, and the target plasmid is 10μg. 4-6 hours after transfection, the medium was replaced with complete medium. After 24, 36, 48, and 60 hours, the supernatant was collected to obtain virus solution.

2. Whole exon sequencing

Tissue samples were extracted with genomic DNA kit DNA, and dissolved in elution buffer (EB) at a concentration of at least 12.5 ng/μL. Test the quality of DNA, take qualified DNA samples greater than 1μg for library construction, and then use Illumina HiSeq X10 for double-end sequencing, with an average coverage of about 130×.

3. Detection of cell viability

Cell viability was detected with CellTiter-Glo (CTG) kit. Incubate the treated 96-well plate cells at room temperature for 30 minutes. The CTG reagent is diluted 4 times with PBS. Add 100 μL of diluted CTG reagent to each well of the cells. Place on a shaker for 2 minutes at room temperature in the dark to mix the cells. After 10 minutes, the luminescence intensity was detected with a microplate reader.

4. Cell cycle detection

The cultured cells were prepared as a single cell suspension, washed with PBS and fixed with 75% ethanol at -20°C, washed with PBS after 24 hours, added RNase A, mixed and incubated at 37°C for 30 minutes, then added propidium iodide (propidiumiodide, PI) DNA staining, after 30 minutes incubation at room temperature in the dark, using flow cytometry.

Example 1

DEPDC5 gene mutation in 40 patients with gastrointestinal stromal tumors (GISTs), 17.5% of GISTs contain DEPDC5 gene mutation

Experimental steps:

1) Extract genomic DNA from patients' tumor and normal tissue samples;

2) Whole exome sequencing of genomic DNA;

3) Sanger sequencing, fluorescent quantitative PCR (qPCR), single nucleotide polymorphism (SNP) genomic analysis, and in situ immunofluorescence hybridization (FISH) were performed on the DNA of patients with mutations.

The results are shown in Figure 1(a-f). The results show that 17.5% of patients with gastrointestinal stromal tumors contain DEPDC5 gene mutations.

Example 2

Once GIST acquires the DEPDC5 gene mutation, the mutation is always accompanied by the evolution of GIST

Experimental steps:

1) SNP genomic analysis of the primary focus, metastatic focus and normal tissues of GIST patients;

2) Perform Sanger sequencing to verify the DNA of the primary, metastatic and normal tissues of patients with mutations;

3) Use the patient's GIST tumor tissue to establish a cell line (GIST882), continuously perform xenotransplantation on nude mice, or select multiple generations of different drugs for continuous SNP whole genome chip analysis;

4) Extract RNA from tumor tissues at different stages of GIST, and then perform quantitative PCR to detect the relative expression of DEPDC5 gene after reverse transcription.

The results are shown in Figure 2(a-e). The results showed that once the gastrointestinal stromal tumor acquired DEPDC5 gene mutation, the mutation was always accompanied by the evolution of gastrointestinal stromal tumor.

Example 3

Compare DEPDC5 gene mutations in GIST and other sarcomas

Experimental steps:

Statistics of the total exon sequencing data of 40 patients with GISTs and the DEPDC5 gene mutation in 255 other sarcomas of TCGA database were compared and compared.

The results are shown in Figure 3. The results show that the mutation frequency of the DEPDC5 gene in gastrointestinal stromal tumors is significantly higher than other sarcomas.

Example 4

DEPDC5 gene deletion promotes tumor growth of GIST cells (in vitro and in vivo experiments)

Experimental steps:

1) Construction of foreign DEPDC5 gene expression vector;

2) Infect GIST882 cells after packaging lentivirus;

3) Observe the cells after 6 days, and use CellTiter-Glo (CTG) kit to detect cell viability;

4) Immunoblotting staining for proliferating cell nuclear antigen (PCNA) to detect cell proliferation;

5) Flow cytometry detects the proportion of each period of the cell cycle, and then reflects cell proliferation;

6) Inject cells under the skin of nude mice for xenotransplantation, and regularly observe and detect tumor growth;

7) Hematoxylin-eosin (HE) staining of xenograft tumors.

The results are shown in Figure 4(a-g). The results show that the deletion of the DEPDC5 gene promotes the growth of gastrointestinal stromal tumor cells.

Example 5

DEPDC5 gene knockout promotes GIST cell growth

Experimental steps:

1) Design two sgRNAs targeting DEPDC5;

2) Construction of Lenti CRISPR v2 gene knockout vector;

3) Infect GIST430 cells after packaging lentivirus;

4) Regularly detect cell viability with CTG kit;

5) PCNA staining to detect cell proliferation by immunoblotting;

6) Flow cytometry detects the proportion of each phase of the cell cycle, and then reflects cell proliferation.

The results are shown in Figure 5(a-c). The results show that the knockout of the DEPDC5 gene promotes the growth of gastrointestinal stromal tumor cells.

Example 6 Inactivation of DEPDC5 in GISTs activates the mTORC1 signaling pathway and promotes cell growth

Experimental steps:

1) Infect GIST882 cells after packaging DEPDC5 gene expression vector lentivirus;

2) Extract cellular RNA samples, and perform gene set enrichment analysis (GSEA) on the data after RNA-seq sequencing;

3) Infect the GIST-T1 and GIST430 cells after packaging the DEPDC5 gene knockout vector lentivirus;

4) Extract the protein samples of GIST882, GIST-T1 and GIST430 cells after lentivirus infection respectively, and detect the expression level of mTOR signaling pathway-related proteins by immunoblotting.

The results are shown in Figure 6(a-c). The results indicate that the inactivation of DEPDC5 in gastrointestinal stromal tumors activates the mTORC1 signaling pathway and promotes cell growth.

Example 7 DEPDC5 regulates the sensitivity of GISTs to KIT inhibitors, DEPDC5 inactivated GIST is effective for the combination of KIT inhibitors and mTOR inhibitors

Experimental steps:

2) Treat GIST882 cells with a gradient concentration of imatinib (Imatinib), and check the cell viability by CTG kit 6 days later;

3) Treat GIST882 cells with a gradient concentration of Imatinib, extract the treated GIST882 cell protein samples after 4 hours, and detect the expression levels of mTOR signaling pathway-related proteins by immunoblotting;

4) GIST882 cells were treated with gradient concentration of imatinib and everolimus, and the cell viability was detected by CTG kit 6 days later;

5) Infect GIST430 cells after packaging DEPDC5 gene knockout vector lentivirus;

6) Treat GIST430 cells with a gradient concentration of Imatinib, and check the cell viability by CTG kit 3 days later;

7) GIST430 cells were treated with gradient concentration of Imatinib and Everolimus respectively, and the cell viability was detected by CTG kit 3 days later.

The results are shown in Figure 7(af). The results show that DEPDC5 regulates the sensitivity of gastrointestinal stromal tumors to KIT inhibitors, and that DEPDC5 inactivated gastrointestinal stromal tumors are effective in combination with KIT inhibitors and mTOR inhibitors .

Claims

A use of DEPDC5 gene, mRNA, cDNA, or protein or its detection reagent, characterized in that (i) is used as a marker for detecting primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor; And/or (ii) for preparing a diagnostic reagent or kit for detecting primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.
A diagnostic kit for detecting the risk of recurrence and metastasis of primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors, characterized in that the kit contains a container, and the container contains detection DEPDC5 gene, mRNA, cDNA, or protein detection reagents; and labels or instructions indicating that the kit is used to detect primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors .
A method for detecting primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor, characterized in that the method includes:

a) Provide test samples from subjects;

b) Detect the expression level of DEPDC5 protein in the test sample and/or the mutation status of DEPDC5 protein; and

c) Compare the expression level of DEPDC5 protein measured in step b) with the control,

Compared with the control, the expression level of DEPDC5 protein in the sample is lower than the reference value, indicating that the probability of the subject suffering from gastrointestinal stromal tumor or metastatic gastrointestinal stromal tumor is higher than that of the general population ( Control group); or

If the DEPDC5 protein contains one or more amino acid mutation sites selected from the following group, it indicates that the subject has a higher risk of primary gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors than the general population ( Control group);

p.P27fs;

p.Q783*;

The corresponding promoter and translation start site of the DEPDC5 protein are deleted;

Among them, the amino acid position number is based on the wild-type human DEPDC5 protein sequence (NP_055477).
A method for determining a treatment plan is characterized by comprising:

a) Provide test samples from subjects;

b) Detect the expression level of DEPDC5 protein in the test sample and/or the mutation status of DEPDC5 protein; and

c) Determine the treatment plan based on the expression level of DEPDC5 protein in the sample and/or the mutation status of DEPDC5 protein.
The use of the DEPDC5 gene, or its protein, or its agonist, characterized in that it is used to prepare a medicament for preventing and/or treating primary gastrointestinal stromal tumor or advanced gastrointestinal stromal tumor.
A pharmaceutical composition characterized by comprising:

(a) DEPDC5 gene, or its protein, or its agonist;

(b) Other drugs to prevent and/or treat primary gastrointestinal stromal tumors or metastatic gastrointestinal stromal tumors; and

(c) A pharmaceutically acceptable carrier.
A medicine box is characterized by comprising:

(a1) The first container, and the DEPDC5 gene, or its protein, or its agonist, or a drug containing the DEPDC5 gene, or its protein, or its agonist, located in the first container;

(b1) a second container, and other medicines for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors located in the second container, or containing other preventive and/or Drugs for treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors.
Use of the pharmaceutical composition according to claim 6 or the kit according to claim 7, characterized in that it is used for the prevention and/or treatment of primary gastrointestinal stromal tumor or advanced gastrointestinal tract Stromal tumor.
An in vitro non-therapeutic method for inhibiting the growth or proliferation of gastrointestinal stromal tumors, comprising the steps of: culturing gastrointestinal stromal tumor cells in the presence of the DEPDC5 gene, or its protein, or its agonist, thereby inhibiting the stomach Intestinal stromal tumor cells grow or proliferate.
A method for screening candidate compounds for preventing and/or treating primary gastrointestinal stromal tumors or advanced gastrointestinal stromal tumors, characterized in that the method comprises the steps of:

(a) In the test group, add the test compound to the cell culture system, and observe the expression level and/or activity of DEPDC5 in the cells of the test group; in the control group, do not add the test in the same cell culture system Compound, and observe the expression level and/or activity of DEPDC5 in the cells of the control group;

Among them, if the DEPDC5 expression and/or activity of the cells in the test group is higher than that of the control group, it indicates that the test compound is a preventive and/or therapeutic treatment for primary gastrointestinal tract that promotes the expression and/or activity of DEPDC5 Candidate compounds for stromal tumors or advanced gastrointestinal stromal tumors.