Application of PKMYT1 in prognosis judgment and treatment of pancreatic cancer

Abstract

The invention relates to application of PKMYT1 in diagnosis and treatment of pancreatic cancer. Specifically, the invention provides application of the PKMYT1 gene, mRNA, cDNA or protein or a detection reagent thereof in pancreatic cancer prognosis, and the PKMYT1 gene or a protein inhibitor thereof can effectively treat pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of oncology and prognosis. More specifically, the present invention relates to the application of PKMYT1 in the prognosis and treatment of pancreatic cancer. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a common digestive system malignancy, accounting for more than 90% of pancreatic cancer. Due to the lack of effective diagnosis and treatment, the 5-year survival rate of PDAC is less than 12%. It is predicted that it will become the third most lethal tumor in the world in 2030. Usually, PDAC is already in the advanced stage when it is diagnosed and cannot be surgically removed. Chemotherapy is the main treatment strategy, such as gemcitabine, 5-fluorouracil, and conjugated albumin paclitaxel, but the effect is minimal. In most cases, it is palliative, with the purpose of alleviating disease-related symptoms and prolonging life. The genomic landscape analysis of PDAC provides a rich source of information for identifying therapeutic targets, among which the targeting of KRAS G12C and BRCA mutants has made effective research progress. Unfortunately, only a small number of PDAC patients carry KRAS p.G12C (about 1%-2%) or BRCA (<9%) mutations. Therefore, finding new strategies for the treatment of pancreatic cancer is a topic that needs to be explored urgently. CRISPR / Cas9 screening is an efficient means of identifying genes essential for tumor growth and proliferation, and is an important strategy for exploring non-mutated targets. Therefore, there is an urgent need to use whole genome screening technology to screen pancreatic cancer for functional loss, explore new candidate target genes for pancreatic cancer treatment, and identify and verify the importance of PKMYT1 for pancreatic cancer proliferation and survival. Summary of the invention

[0003] The purpose of the present invention is to use whole genome screening technology to screen pancreatic cancer for functional loss, explore new candidate target genes for pancreatic cancer treatment, identify and verify the importance of PKMYT1 for the proliferation and survival of pancreatic cancer, and find that PKMYT1 has significant prognostic relevance, and functional intervention of PKMYT1 can effectively inhibit the proliferation ability of PDAC.

[0004] In a first aspect, the present invention provides a use of a PKMYT1 gene, mRNA, cDNA, or protein or a detection reagent thereof, (i) as a marker for determining the prognosis and survival period of a pancreatic cancer patient, and / or (ii) for preparing a diagnostic reagent or kit for determining the prognosis and survival period of a pancreatic cancer patient.

[0005] In another preferred example, the pancreatic cancer includes grade 4 pancreatic cancer.

[0006] In another preferred embodiment, the diagnostic reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

[0007] In another preferred embodiment, the protein includes a full-length protein or a protein fragment.

[0008] In another preferred embodiment, the PKMYT1 gene, mRNA, cDNA, or protein is derived from mammals, preferably from rodents (such as mice, rats), primates and humans, and more preferably from patients diagnosed with pancreatic cancer.

[0009] In another preferred embodiment, the PKMYT1 gene, mRNA, cDNA, or protein is derived from a pancreatic cancer patient.

[0010] In another preferred example, the accession number of the PKMYT1 gene is NC_000016.

[0011] In another preferred embodiment, the accession number of the PKMYT1 mRNA is NM_004203.5.

[0012] In another preferred example, the accession number of the PKMYT1 protein is NP_004194.3.

[0013] In another preferred embodiment, the detection is tissue sample detection.

[0014] In another preferred embodiment, the detection includes immunohistochemistry, immunoblotting and fluorescence quantitative PCR detection.

[0015] In another preferred embodiment, the detection includes measuring pancreatic cancer tissue or general pancreatic tissue samples.

[0016] In another preferred embodiment, the general pancreatic tissue includes paracancerous tissue.

[0017] In another preferred embodiment, the detection reagent includes a specific antibody for PKMYT1, a specific binding molecule for PKMYT1, a specific amplification primer, a probe or a chip.

[0018] In another preferred embodiment, the PKMYT1 protein or its specific antibody or specific binding molecule is coupled with or carries a detectable label.

[0019] In another preferred embodiment, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0020] In another preferred embodiment, the specific antibody against PKMYT1 is a monoclonal antibody or a polyclonal antibody.

[0021] In another preferred embodiment, the PKMYT1 protein also includes derivatives of the PKMYT1 protein.

[0022] In another preferred embodiment, the derivatives of the PKMYT1 protein include modified PKMYT1 protein, protein molecules whose amino acid sequences are homologous to natural PKMYT1 protein and have the activity of natural PKMYT1 protein, and fusion proteins containing the amino acid sequence of PKMYT1 protein.

[0023] In another preferred embodiment, the modified PKMYT1 protein is a PEGylated PKMYT1 protein.

[0024] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to the natural PKMYT1 protein and has the activity of the natural PKMYT1 protein" refers to a protein molecule whose amino acid sequence has ≥85% homology, preferably ≥90% homology, more preferably ≥95% homology, and optimally ≥98% homology with the PKMYT1 protein; and has the activity of the natural PKMYT1 protein.

[0025] A second aspect of the present invention provides a diagnostic kit for determining the prognosis and survival period of pancreatic cancer patients, the kit comprising a container containing a detection reagent for detecting PKMYT1 gene, mRNA, cDNA, or protein; and a label or instructions, the label or instructions indicating that the kit is used to determine the prognosis and survival period of pancreatic cancer patients.

[0026] In another preferred example, the judgment includes prejudgment (prediction).

[0027] In another preferred embodiment, the detection reagent for detecting PKMYT1 gene, mRNA, cDNA, or protein comprises:

[0028] (a) Anti-PKMYT1 protein specific antibodies; and / or

[0029] (b). Specific primers for specific amplification of PKMYT1 mRNA or cDNA.

[0030] In another preferred embodiment, the detection is tissue sample detection.

[0031] In another preferred embodiment, the label or instructions state the following:

[0032] When the ratio of the expression level of PKMYT1 E1 in the pancreatic cancer cells or tissues of the test subject to the expression level of PKMYT1 E2 in the general pancreatic cells or tissues is ≥2, it indicates that the probability of poor prognosis and shortened survival of the test subject is higher than that of the general population;

[0033] Wherein, the E2 is the expression level of PKMYT1 in general pancreatic cells or tissues of the general population.

[0034] A third aspect of the present invention provides a method for determining the prognosis and survival period of a pancreatic cancer patient, the method comprising:

[0035] a) providing a test sample from a subject;

[0036] b) detecting the expression level E1 of PKMYT1 protein in the test sample; and

[0037] c) comparing the expression level E1 of the PKMYT1 protein determined in step b) with that of the control,

[0038] Wherein, compared with the control, the expression level E1 of the PKMYT1 protein in the sample is higher than the reference value, indicating that the probability of poor prognosis and shortened survival of the subject is higher than that of the general population (control group); and / or

[0039] Compared with the control, the expression level of PKMYT1 protein in the sample was lower than the reference value, indicating that

[0040] The subjects had a lower chance of poor prognosis and shortened survival than the general population (control group).

[0041] In another preferred embodiment, the subject is a human or non-human mammal.

[0042] In another preferred embodiment, the test sample is pancreatic cancer cells or tissues.

[0043] In another preferred embodiment, the reference value is a cut-off value.

[0044] In another preferred embodiment, the reference value is the relative expression level of PKMYT1 in the sample.

[0045] In another preferred embodiment, the reference value is 2.

[0046] In another preferred embodiment, the detection step (b) comprises detecting the amount of PKMYT1 mRNA, or the amount of secreted PKMYT1 in serum; and / or detecting the amount of PKMYT1 protein in tissue.

[0047] In another preferred embodiment, the expression level of PKMYT1 protein in the sample is detected by fluorescent quantitative PCR or immunohistochemistry.

[0048] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0049] A fourth aspect of the present invention provides a method for determining a treatment regimen, comprising:

[0050] a) providing a test sample from a subject;

[0051] b) detecting the expression level of PKMYT1 protein in the test sample; and

[0052] c) determining a treatment regimen based on the expression level of PKMYT1 protein in the sample.

[0053] In another preferred embodiment, the subject is a human or non-human mammal.

[0054] In another preferred embodiment, when the expression level of PKMYT1 protein in the sample tissue is higher than the reference value, it indicates that the subject's probability of poor prognosis and shortened survival is higher than that of the general population (control group population), and the treatment regimen includes PKMYT1 inhibitor therapy, and a therapy combining PKMYT1 inhibitor with other drugs for the prevention and / or treatment of pancreatic cancer.

[0055] In another preferred embodiment, the PKMYT1 inhibitor therapy, or the combination therapy of the PKMYT1 inhibitor and other drugs for treating pancreatic cancer is selected from the following group:

[0056] PKMYT1 inhibitor therapy: antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof;

[0057] Other drug therapies for pancreatic cancer:

[0058] Gemcitabine, albumin-bound (nab) paciltaxel, FOLFIRINOX combination drug, or a combination thereof.

[0059] In another preferred embodiment, the PKMYT1 inhibitor is selected from the following group: RP-6306, Dasatinib, PD0166285, or a combination thereof.

[0060] In another preferred embodiment, when the subjects have a poor prognosis and a shorter survival rate than the general population (control group), the treatment regimen also includes PKMYT1 inhibitor therapy, or the combination of PKMYT1 inhibitor and other drugs for preventing and / or treating pancreatic cancer.

[0061] In another preferred embodiment, the other drugs for preventing and / or treating pancreatic cancer include gemcitabine, albumin-bound (nab) paciltaxel, and FOLFIRINOX combination drug.

[0062] A fifth aspect of the present invention provides a use of a PKMYT1 gene or protein inhibitor thereof for preparing a composition or preparation for (a) inhibiting the growth and / or proliferation of pancreatic cancer cells; and / or (b) preventing and / or treating pancreatic cancer.

[0063] In another preferred embodiment, the PKMYT1 gene or its protein inhibitor is selected from the following group: antibodies, small molecule compounds, microRNA, siRNA, shRNA, CRISPR reagents, or a combination thereof.

[0064] In another preferred embodiment, the CRISPR reagent comprises sgRNA and Cas protein.

[0065] In another preferred embodiment, the inhibitor comprises shRNA, a CRISPR reagent comprising sgRNA and a Cas protein.

[0066] In another preferred embodiment, the shRNA targets the positive strand, and the target sequence corresponding to the positive strand is CCATCCCTTATAGGTTAATAT. .

[0067] In another preferred embodiment, the inhibitor includes a substance that inhibits the expression of PKMYT1 gene or its protein.

[0068] In another preferred embodiment, the inhibitor includes an inhibitor that inhibits the expression of PLK1.

[0069] In another preferred embodiment, the inhibitor includes an inhibitor that inhibits the function and expression of p53, or an inhibitor that impairs the function of the G1 / S cycle checkpoint.

[0070] In another preferred embodiment, the composition or preparation is also used for one or more purposes selected from the following group:

[0071] (i) Inducing G2 / M arrest of the cell cycle, accumulating a large amount of pan-nuclear DNA damage, and producing an apoptotic phenotype;

[0072] (ii) produces an apoptotic and cycle arrest phenotype consistent with PKMYT1 inhibition;

[0073] (iii) Increasing PRKDC phosphorylation and activating it, inhibiting the tumor suppressor effect of PKMYT1 depends on the function of PRKDC.

[0074] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0075] In another preferred embodiment, the composition comprises a therapeutically effective amount of an inhibitor of PKMYT1 gene or its protein, and a pharmaceutically acceptable carrier.

[0076] In another preferred embodiment, the drug is administered by a route selected from the group consisting of oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, rectal irrigation, nasal spray, oral spray, topical skin administration or systemic transdermal administration.

[0077] In another preferred embodiment, the preparation is selected from the following group: tablets, capsules, injections, granules, and sprays.

[0078] The sixth aspect of the present invention provides a pharmaceutical composition comprising:

[0079] (a1) an inhibitor of the PKMYT1 gene or its protein; and

[0080] (b) a pharmaceutically acceptable carrier.

[0081] In another preferred embodiment, the pharmaceutical composition further comprises:

[0082] (c) Other drugs for the prevention and / or treatment of pancreatic cancer.

[0083] In another preferred embodiment, in the pharmaceutical composition, the content of component (a1) is 1%-99%, preferably, 10%-90%, and more preferably, 30%-70%.

[0084] In another preferred embodiment, in the pharmaceutical composition, the content of component (c) is 1%-99%, preferably, 10%-90%, and more preferably, 30%-70%.

[0085] In another preferred embodiment, in the pharmaceutical composition, the component (a1) and the optional component (c) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the pharmaceutical composition.

[0086] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection form and an oral dosage form.

[0087] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0088] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0089] A seventh aspect of the present invention provides a medicine kit, comprising:

[0090] (a1) a first container, and an inhibitor of PKMYT1 gene or protein thereof, or a drug containing an inhibitor of PKMYT1 gene or protein thereof, located in the first container.

[0091] In another preferred embodiment, the medicine kit further comprises:

[0092] (b1) a second container, and other drugs for preventing and / or treating pancreatic cancer, or drugs containing other drugs for preventing and / or treating pancreatic cancer, located in the second container.

[0093] In another preferred embodiment, the drug kit further comprises (c1) a third container, and a detection reagent for PKMYT1 located in the third container.

[0094] In another preferred embodiment, the first container, the second container and the third container are the same or different containers.

[0095] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing an inhibitor of the PKMYT1 gene or its protein.

[0096] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing other drugs for preventing and / or treating pancreatic cancer.

[0097] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0098] In another preferred embodiment, the kit further contains instructions.

[0099] In another preferred embodiment, the description records one or more instructions selected from the following group:

[0100] (i) using an inhibitor of the PKMYT1 gene or its protein for (a) inhibiting the growth and / or proliferation of pancreatic cancer cells; and / or (b) preventing and / or treating pancreatic cancer;

[0101] (ii) combining an inhibitor of the PKMYT1 gene or its protein with other drugs for preventing and / or treating pancreatic cancer to (a) inhibit the growth and / or proliferation of pancreatic cancer cells; and / or (b) prevent and / or treat pancreatic cancer;

[0102] (iii) detecting the expression level of PKMYT1 gene or its protein in pancreatic cancer patients, and administering an inhibitor of PKMYT1 gene or its protein to (a) inhibit the growth and / or proliferation of pancreatic cancer cells; and / or (b) prevent and / or treat pancreatic cancer;

[0103] (iv) detecting the expression level of PKMYT1 gene or its protein in pancreatic cancer patients, and simultaneously using an inhibitor of PKMYT1 gene or its protein; and other drugs for preventing and / or treating pancreatic cancer to (a) inhibit the growth and / or proliferation of pancreatic cancer cells; and / or (b) prevent and / or treat pancreatic cancer.

[0104] (v) using an inhibitor of PKMYT1 gene or its protein to (i) induce G2 / M arrest of the cell cycle, accumulation of large amounts of pan-nuclear DNA damage, and an apoptotic phenotype; and / or (ii) produce an apoptotic and cell cycle arrest phenotype consistent with PKMYT1 inhibition; and / or (iii) increase PRKDC phosphorylation and activation, thereby inhibiting the tumor suppressor effect of PKMYT1 that is dependent on PRKDC function;

[0105] (vi) Inhibitors of the PKMYT1 gene or its protein are combined with other drugs for the prevention and / or treatment of pancreatic cancer to (i) induce G2 / M arrest of the cell cycle, accumulate a large amount of pan-nuclear DNA damage, and produce an apoptotic phenotype; and / or (ii) produce an apoptotic and cell cycle arrest phenotype consistent with PKMYT1 inhibition; and / or (iii) increase PRKDC phosphorylation and activation, thereby inhibiting the tumor suppressor effect of PKMYT1 that depends on the function of RKDC.

[0106] The eighth aspect of the present invention provides a use of the pharmaceutical composition described in the sixth aspect of the present invention or the medicine kit described in the seventh aspect of the present invention for (a) inhibiting the growth and / or proliferation of pancreatic cancer cells; and / or (b) preventing and / or treating pancreatic cancer.

[0107] In another preferred embodiment, in the pharmaceutical composition, the effective concentration of the inhibitor of PKMYT1 gene or its protein is 0.0005-500 μM, preferably, 0.05-50 μM, more preferably, 1-10 μM.

[0108] In another preferred embodiment, in the pharmaceutical composition, the effective concentration of the other drugs for preventing and / or treating pancreatic cancer is 0.0005-500 μM, preferably 0.05-50 μM, and more preferably 1-10 μM.

[0109] In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of PKMYT1 gene or its protein; and (b) optionally other drugs for preventing and / or treating pancreatic cancer; and (d) a pharmaceutically acceptable carrier.

[0110] In another preferred embodiment, in the pharmaceutical composition or medicine kit, the inhibitor of PKMYT1 gene or its protein; and (b) optional other drugs for preventing and / or treating pancreatic cancer account for 0.01-99.99wt%, preferably 0.1-90wt%, and more preferably 1-80wt% of the total weight of the pharmaceutical composition or medicine kit.

[0111] A ninth aspect of the present invention provides a method for preventing and / or treating pancreatic cancer, comprising:

[0112] Administering an inhibitor of PKMYT1 gene or its protein to a subject in need; or the pharmaceutical composition according to the sixth aspect of the present invention or the drug kit according to the seventh aspect of the present invention.

[0113] In another preferred embodiment, the subject includes a human or non-human mammal suffering from pancreatic cancer.

[0114] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0115] In another preferred embodiment, the administration dosage of the inhibitor of PKMYT1 gene or protein thereof is 0.05-50000 mg / kg body weight, preferably 2.5-100 mg / kg body weight, and most preferably 5-50 mg / kg body weight.

[0116] In another preferred embodiment, the frequency of administration of the inhibitor of PKMYT1 gene or protein thereof is 1-4 times / day, preferably 2-3 times / day.

[0117] In another preferred embodiment, the interval between two consecutive administrations of the inhibitor of PKMYT1 gene or protein thereof is more than 4 hours, preferably more than 8 hours.

[0118] In another preferred embodiment, the inhibitor of PKMYT1 gene or protein thereof is administered simultaneously or sequentially with other optional drugs for preventing and / or treating pancreatic cancer.

[0119] The tenth aspect of the present invention provides an in vitro non-therapeutic method for inhibiting the growth or proliferation of pancreatic cancer cells, comprising the steps of: culturing pancreatic cancer cells in the presence of a PKMYT1 gene or a protein inhibitor thereof, thereby inhibiting the growth or proliferation of pancreatic cancer cells.

[0120] In another preferred embodiment, the PKMYT1 gene or protein inhibitor is selected from the following group: antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof.

[0121] In another preferred embodiment, the PKMYT1 gene or protein inhibitor is selected from the following group: RP-6306, Dasatinib, PD0166285, or a combination thereof.

[0122] In another preferred embodiment, the pancreatic cancer cells highly express PKMYT1 protein.

[0123] In another preferred embodiment, the method further comprises adding other drugs for preventing and / or treating pancreatic cancer to the culture system of pancreatic cancer cells, thereby inhibiting the growth or proliferation of pancreatic cancer cells.

[0124] In another preferred embodiment, the pancreatic cancer cells are cells cultured in vitro.

[0125] The eleventh aspect of the present invention provides a method for screening candidate compounds for preventing and / or treating pancreatic cancer, the method comprising the steps of:

[0126] (a) In a test group, a test compound is added to the cell culture system, and the expression level (E1) and / or activity (A1) of PKMYT1 in the cells of the test group is observed; in a control group, no test compound is added to the same cell culture system, and the expression level (E0) and / or activity (A0) of PKMYT1 in the cells of the control group is observed;

[0127] Among them, if the expression level (E1) and / or activity (A1) of PKMYT1 in the cells of the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate compound for preventing and / or treating pancreatic cancer that has an inhibitory effect on the expression and / or activity of PKMYT1.

[0128] In another preferred embodiment, the expression level of PKMYT1 is obtained by fluorescence quantitative PCR, high-throughput transcriptome sequencing or immunohistochemistry detection.

[0129] In another preferred embodiment, the method further comprises the steps of:

[0130] (b) For the candidate compound obtained in step (a), further testing its inhibitory effect on the growth or proliferation of pancreatic cancer cells; and / or further testing whether it has a down-regulating effect on the PKMYT1 gene.

[0131] In another preferred embodiment, step (b) includes the following steps: in the test group, the test compound is added to the culture system of pancreatic cancer cells, and the number and / or growth of pancreatic cancer cells is observed; in the control group, the test compound is not added to the culture system of pancreatic cancer cells, and the number and / or growth of pancreatic cancer cells is observed; wherein, if the number or growth rate of pancreatic cancer cells in the test group is lower than that in the control group, it indicates that the test compound is a candidate compound for preventing and / or treating pancreatic cancer that has an inhibitory effect on the growth or proliferation of pancreatic cancer cells.

[0132] In another preferred embodiment, the method comprises step (c): administering the candidate compound determined in step (a) to a mammalian model to determine its effect on the mammal.

[0133] In another preferred embodiment, the mammal is a mammal suffering from pancreatic cancer.

[0134] In another preferred embodiment, the “significantly lower than” means E1 / E0≤1 / 2, preferably ≤1 / 3, and more preferably ≤1 / 4.

[0135] In another preferred embodiment, the “significantly lower than” means A1 / A0≤1 / 2, preferably ≤1 / 3, more preferably ≤1 / 4.

[0136] In another preferred embodiment, the cells include pancreatic cancer cells.

[0137] In another preferred embodiment, the cells are cells cultured in vitro.

[0138] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0139] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 Shown is the identification of the PDAC-dependent gene PKMYT1 by a genome-wide CRISPR loss-of-function screen.

[0141] a. Flowchart of genome-wide CRISPR / Cas9 knockout screening in PDAC cells.

[0142] b. Cumulative frequencies of sgRNAs in the genome-wide screen in Pa-Tu-8988T and YAPC cells at population doublings of 0 and 14. The shift in the curve at population doublings of 14 indicates depletion of a subset of sgRNAs.

[0143] c. Correlation between med.CS values ​​of screening results of library A and library B of GeCKOv2 library.

[0144] d. Correlation between Pa-Tu-8988T and YAPC cell screening results med.CS.

[0145] e. Use different algorithms to analyze the correlation of data.

[0146] f. Venn diagram showing the intersection between the top 1000 (~5%) genes with the lowest med.CS values ​​in the two cell lines.

[0147] g. DAVIDE analysis showed that the 1% most important genes in PDAC screening were significantly enriched in a variety of basic cellular biological activities.

[0148] h. The med.CS value ranking of the whole genome screening results of Pa-Tu-8988T, YAPC, GIST-T1 and GIST430 cells. The red dots represent the PKMYT1 gene. The blue dots represent the SDHC gene, and the green dots represent the COX7B gene.

[0149] Figure 2 It was shown that PKMYT1 was highly expressed in PDAC and had significant clinical significance.

[0150] a. Immunohistochemical analysis of 2 formalin-fixed paraffin-embedded surgical specimens from clinical PDAC patients showed high expression of PKMYT1.

[0151] b. Expression of PKMYT1 in different subtypes of pancreatic cancer tissues. Grade 1, well-differentiated (low grade); Grade 2, moderately differentiated (intermediate grade); Grade 3, poorly differentiated (high grade); Grade 4, undifferentiated (high grade). Data from TCGA database.

[0152] c. The GEPIA tool was used to analyze the expression of PKMYT1 in human pancreatic cancer tissues (n=179) and non-cancerous tissues (n=171) from the TCGA database (the vertical axis is the logarithmic value result).

[0153] d. The data were grouped according to the median value of PKMYT1 expression level, and the overall survival curve was analyzed by Kaplan-Meier. PDAC patients with high PKMYT1 expression had a poor prognosis. Analysis data sources: GSE71729 (p = 0.0386, hazard ratio [HR] = 1.595 [1.025-2.481, 95% confidence interval (95% CI)]), TCGA database pancreatic cancer samples (p = 0.0444, HR = 1.535 [1.011-2.33295% CI]), GSE21501 (p = 0.0165, HR = 1.586 [1.1.119-3.077, 95% CI]).

[0154] e. Frequency of PKMYT1 genomic amplification in the database. The number of cases in the cohort is shown above the column.

[0155] Figure 3 We showed that PKMYT1 knockout inhibited the growth and proliferation of PDAC in vivo cell models and in vitro xenograft models.

[0156] a. Immunoblotting experiments verified the PKMYT1 knockout efficiency.

[0157] b. CellTiter-Glo experiment evaluated the effect of PKMYT1 knockout on cell survival. The activity of pancreatic cancer cell lines and primary cultured cell lines was reduced after PKMYT1 knockout.

[0158] c. Plate cloning experiments show that PKMYT1 functional loss inhibits the cloning ability and proliferation ability of PDAC cells. The left picture is a statistical chart, and the right picture is a representative picture.

[0159] d. Soft agar experiments showed that loss of PKMYT1 function would lead to decreased anchorage-independent proliferation of PDAC cells. The left picture is a statistical chart, and the right picture is a representative picture.

[0160] Eg. Mouse subcutaneous tumor formation experiments showed that PKMYT1 deficiency attenuated the growth of Pa-Tu-8988T, YAPC and primary cell PDAC-CN1 xenograft tumors in nude mice. Shown are the growth curves (e), representative images (f) and tumor weights (g) of transplanted tumors. The mean standard error was determined by 6 replicates.

[0161] h. Flow cytometric analysis revealed that PKMYT1 knockout would alter cell cycle progression, increase the number of cells in the G2 / M phase, and cause cell cycle arrest.

[0162] i. Immunofluorescence experiments showed that PKMYT1 knockdown induced pan-nuclear DNA damage (as measured by the level of γH2A.X).

[0163] j. Flow cytometric analysis showed that PKMYT1 deficiency increased the levels of early and late apoptosis of cells.

[0164] Ctrl, control group. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean was determined by 3 replicates unless otherwise noted.

[0165] Figure 4 showed that PKMYT1 knockout inhibited the growth and proliferation of PDAC.

[0166] a. Immunoblotting experiments were performed to verify the PKMYT1 knockout efficiency in four pancreatic cancer cell lines (KP4, MiaPaCa2, SNU-324 and BxPC3) and one primary cultured cell line (PDAC-CN2).

[0167] b. CellTiter-Glo experiment evaluated the effect of PKMYT1 knockout on cell survival. The activity of pancreatic cancer cell lines and primary cultured cell lines was reduced after PKMYT1 knockout.

[0168] c. Western Blotting showed that knocking out PKMYT1 could reduce CDK1 phosphorylation and increase the level of DNA damage (represented by the level of γH2A.X), but did not affect PCNA expression.

[0169] d. Flow cytometric analysis revealed that PKMYT1 elimination would alter cell cycle progression, increase the number of cells in the G2 / M phase, and cause cell cycle arrest. Figure 3 h Data represent Fig.

[0170] e. Flow cytometry analysis revealed that PKMYT1 deficiency increases the levels of early and late apoptosis in cells. Figure 3 j Data represent Fig.

[0171] f. Gene set enrichment analysis of transcriptome data after PKMYT1 knockout revealed that gene sets related to mitosis and G2 / M checkpoint were significantly enriched.

[0172] g. Effect of PKMYT1 knockout on gemcitabine sensitivity.

[0173] Ctrl, control group. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean after 3 replicates.

[0174] Figure 5 The PKMYT1 inhibitor RP-6306 was shown to inhibit tumor growth and proliferation in multiple PDAC cell models in vitro.

[0175] a. RP-6306 can effectively inhibit the activity of PDAC expressing PKMYT1.

[0176] b. RP-6306 treatment increased the hyperphosphorylation level of PKMYT1 in PDAC cells (migration rate slowed down) and reduced its kinase activity (characterized by the phosphorylation degree of CDK1) in a dose-dependent manner.

[0177] c. RP-6306 treatment increases DNA damage accumulation in PDAC in a dose- and time-dependent manner.

[0178] d. Flow cytometry analysis demonstrated that RP-6306 treatment could cause cell cycle arrest, and the degree of arrest was related to the drug dose.

[0179] e. Flow cytometry analysis demonstrated that RP-6306 treatment could increase cell apoptosis in a dose-dependent manner.

[0180] Ctrl, control group. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean after 3 replicates.

[0181] Figure 6 The PKMYT1 inhibitor RP-6306 was shown to inhibit the in vivo growth of cell line-derived xenografts as well as xenografts derived from tumor patients without producing significant toxic side effects.

[0182] af. Subcutaneous tumor formation experiments in mice showed that PKMYT1 deficiency attenuated the growth of xenograft tumors CDX (ac) and xenograft tumors PDAX-PDX2 (df) derived from tumor patients in nude mice with Pa-Tu-8988T cell line. Shown are the growth curves of transplanted tumors (a, d), representative images (b, e) and mouse body weights (c, f). In the CDX 5 mg / kg group, one mouse experienced tumor regression and disappeared. (CDX is the mean standard error of 6 repeated measurements, and PDAC-PDX2 is the mean standard error of 8 repeated measurements).

[0183] gh.RP-6306 administration in vivo does not increase liver damage in mice. The figure shows the activities of alanine aminotransferase and aspartate aminotransferase. The mean standard error is the average of 6 replicates.

[0184] ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test.

[0185] Figure 7 It was shown that the PKMYT1 inhibitor RP-6306 can inhibit the in vivo growth and proliferation of cell line-derived xenograft tumors and xenograft tumors derived from tumor patients without producing obvious toxic side effects.

[0186] PR-6306 treatment did not cause significant differences in blood routine indices in the Pa-Tu-8988T xenograft model (a) and PDX model (b). WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin content; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; PDW, platelet distribution width; MPV, mean platelet volume; P-LCR, platelet-large cell ratio; PCT, platelet volume; NRBC, nucleated red blood cell; NEUT, neutrophil; LYMPH, lymphocyte; MONO, monocyte; EO, eosinophil; BASO, basophil.

[0187] ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean after 6 replicates.

[0188] Figure 8 We show that the PKMYT1 inhibitor PD0166285 can suppress proliferation in multiple PDAC models and produce apoptotic and cycle arrest phenotypes consistent with PKMYT1 inhibition.

[0189] a. Western Blotting experiments showed that PD0166285 treatment in PDAC cells reduced the phosphorylation level of threonine 14 of CDK1 in a dose- and time-dependent manner.

[0190] b. PD0166285 can effectively inhibit PDAC activity.

[0191] c. PD0166285 treatment increased DNA damage accumulation in PDAC cells in a dose-dependent manner.

[0192] d. Flow cytometry analysis demonstrated that PD0166285 treatment could increase cell apoptosis in a dose-dependent manner.

[0193] e. Flow cytometry analysis demonstrated that PD0166285 treatment could cause cell cycle arrest, and the degree of arrest was related to the drug dose.

[0194] ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean after 3 replicates.

[0195] Fig. 9 We show that the PKMYT1 inhibitor Dasatinib inhibits proliferation in multiple PDAC models and produces apoptotic and cycle arrest phenotypes consistent with PKMYT1 inhibition.

[0196] a. Western Blotting experiments showed that Dasatinib treatment in PDAC cells reduced the phosphorylation level of threonine 14 of CDK1 in a dose-dependent manner.

[0197] b.Dasatinib can effectively inhibit the activity of PDAC.

[0198] c. Flow cytometry analysis demonstrated that Dasatinib treatment could increase cell apoptosis.

[0199] d. Flow cytometry analysis demonstrated that Dasatinib treatment can cause cell cycle arrest

[0200] ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean after 3 replicates.

[0201] Fig.10 We show that PKMYT1 interacts with PLK1 and PRKDC in PDAC and that PLK1 mediates the oncogenic role of PKMYT1 in pancreatic cancer.

[0202] ab. Flag-PKMYT1 was introduced into Pa-Tu-8988T, and IP experiments were performed using anti-Flag antibodies. The IP products were subjected to SDS-PAGE electrophoresis and then Coomassie staining (a). Mass spectrometry proteomics identified a variety of proteins that interact with PKMYT1 protein, and the table shows the protein data with high confidence. The results showed that PRKDC and PLK1 were the most enriched binding proteins (b).

[0203] c. Western Blotting experiments showed that RP-6306 treatment would downregulate the protein expression level of PLK1 and increase the phosphorylation of serine 2056 of PRKDC.

[0204] d. Western Blotting experiments showed that the protein expression level of PLK1 was downregulated and the phosphorylation level of serine 2056 of PRKDC was upregulated after PKMYT1 knockout.

[0205] e. Western Blotting experiments demonstrated that the introduction of PKMYT1 would increase the downregulation of PLK1 protein expression level.

[0206] f. After the control or PKMYT1 gene knockout plasmids were introduced into PDAC cells, they were treated with 10 μM cycloheximide (CHX) at 0, 3, and 6 h, and then Western Blotting experiments were performed to detect changes in the protein expression level of PLK1.

[0207] g. After the control or PKMYT1 gene knockout plasmids were introduced into PDAC cells, they were treated with 10 μM CHX at 0 h and 4 h, and then Western Blotting experiments were performed to detect the changes in the protein expression level of PLK1.

[0208] h. CellTiter-Glo experiment verified that the cell activity of PDAC cells decreased after PLK1 knockout.

[0209] i. Flow cytometric analysis revealed that PLK1 deficiency increased the levels of early and late apoptosis in cells.

[0210] j. Flow cytometric analysis revealed that PLK1 knockout would alter cell cycle progression, increase the number of cells in the G2 / M phase, and cause cell cycle arrest.

[0211] k. CellTiter-Glo activity assay was performed after overexpression of PKMYT1-sgR, PLK1, or GFP in YAPC cell lines transduced with sgRNA targeting PKMYT1 or control sgRNA. The results showed that high expression of PLK1 significantly attenuated the proliferation inhibition caused by PKMYT1 knockout in cells.

[0212] Ctrl, control group. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean after 3 replicates.

[0213] Fig.11 It was shown that in PDAC, in addition to its classical kinase function, PKMYT1 can also interact with PLK1 and regulate its expression.

[0214] ab.CTG experiments showed that in YAPC cells, compared with the control group, complementation of PKMYT1 in PKMYT1 knockout cell lines alleviated cell proliferation inhibition to a certain extent, but complementation of the PKMYT1-N238A mutant with missing kinase activity could not restore cell survival. Ctrl, control group. ns, no statistical significance; **, p<0.01. One-tailed t test. Mean standard error of 3 replicates.

[0215] c. Cells were transduced with Flag-PKMYT1 or Flag-PLK1, and then the cell lysates were immunoprecipitated with anti-Flag antibodies, followed by immunoblotting with antibodies. The experiment demonstrated that PKMYT1 and PLK1 interacted in Pa-Tu-8988T (left). PRKDC interacted with PLK1 in PDAC cells (right).

[0216] d. Analysis of PKMYT1 and PLK1 expression in the Ualcan database revealed a strong correlation between the two.

[0217] e. Expression of PLK1 in different subtypes of pancreatic cancer tissues.

[0218] f. PLK1 knockout hardly induces DNA damage in PDAC cells.

[0219] g. Treatment with the PLK1 inhibitor Onvansertib does not induce DNA damage in PDAC cells.

[0220] h. In vitro kinase assay verified that PKMYT1 can directly phosphorylate PLK1.

[0221] ij.CTG experiments showed that in YAPC cells, compared with the control group, the restoration of PLK1 protein with persistent phosphorylation at position 217 in PKMYT1 knockout cell lines improved cell proliferation inhibition to a certain extent. sgCtrl, control group. One-tailed t-test. Mean standard error of 3 replicates.

[0222] Fig.12It was shown that PKMYT1 inhibition in PDAC increased the expression of TNFAIP3, BIRC3, and EGR1, and TNFAIP3 and BIRC3 could mediate the regulation of PKMYT1 on PLK1 expression.

[0223] ab. The expression of genes encoding ubiquitin ligases TNFAIP3, BIRC3, and transcription factor EGR1 was upregulated in Pa-Tu-8988T and YAPC cells after PKMYT1 knockout (a) or RP-6306 treatment (b). The vertical axis is the TPM value of these three genes detected in transcript sequencing (RNA-seq).

[0224] cd. Western Blotting experiments demonstrated that the expression of TNFAIP3, BIRC3 and EGR1 proteins were upregulated in Pa-Tu-8988T and YAPC cells with PKMYT1 knockout (c) or RP-6306 treatment (d).

[0225] e. Knockout of TNFAIP3 (left) and BIRC3 (right) attenuates the downregulation of PLK1 caused by PKMYT1 inhibition.

[0226] f. Ubiquitin ligase TNFAIP3 can increase the ubiquitination of PLK1 in vitro.

[0227] Fig.13 It was shown that PKMYT1 inhibition in PDAC increased PRKDC phosphorylation and activation, and the tumor suppressor effect produced by inhibiting PKMYT1 was dependent on the function of PRKDC.

[0228] a. Immunoprecipitation experiments demonstrated the interaction between PKMYT1 and PRKDC in Pa-Tu-8988T and YAPC. Cells were transduced with a PKMYT1 plasmid with a FLAG tag (NF-PKMYT1), and then cell lysates were immunoprecipitated with anti-Flag antibodies, followed by immunoblotting experiments using antibodies.

[0229] bc. PRKDC inhibitors NU7441 (b) and NU7026 (c) blocked the accumulation of γH2A.X induced by RP-6306 treatment in a dose-dependent manner.

[0230] d. CTG experiments showed that knockdown of PRKDC increased the resistance of PDAC cells to the RP-6306 inhibitor. The mean standard error was obtained after 3 replicates.

[0231] ef. Combination index analysis and isobologram analysis showed that there was a clear antagonistic effect between PRKDC inhibitors NU7441 (e) and NU7026 (f) and PKMYT1 inhibitors.

[0232] Fig.14 We show that TP53 mutation status influences PDAC's dependence on PKMYT1.

[0233] a. PKMYT1 expression in PDAC is significantly correlated with the p53 pathway.

[0234] b. TP53 mutation status in PDAC is associated with PKMYT1 expression.

[0235] c. Sanger sequencing verified that Trp53 sgRNA produced multiple insertion / deletion mutations in KC cell genome editing, and Trp53 was inactivated in KPC. The heteroallelic model was successfully established.

[0236] d. CTG experiments showed that PKMYT1 knockout did not basically inhibit the survival of cells with normal p53 function (KC, BJ and GIST430 cell lines), but inhibited the survival of KPC cells lacking TP53 (Trp53).

[0237] e. Sanger sequencing verified that Pkmyt1 sgRNAs produced multiple insertion and deletion mutations in KC and KPC cell genome editing.

[0238] f. The results of CTG experiments showed that KPC#10 cell line was more sensitive to RP-6306 than KC cells.

[0239] g. Plate cloning experiments show that Pkmyt1 functional loss inhibits the cloning ability and proliferation ability of PDAC cells. The left figure is a statistical graph, and the right figure is a representative graph. (Unpaired t-test, 3 repeated determinations of the mean standard error.

[0240] h. Mouse subcutaneous tumor formation experiments showed that Pkmyt1 deficiency weakened the growth of KPC cell xenografts in nude mice, but had little effect on the subcutaneous tumor formation ability of KC cells. Shown are the growth curves, tumor weights and representative images of transplanted tumors of KC and KPC cell lines. The mean standard error was determined by 6 replicates.

[0241] Ctrl, control group. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Unpaired t-test, standard error of the mean was determined by 3 replicates unless otherwise noted. DETAILED DESCRIPTION

[0242] After extensive and in-depth research, the inventor unexpectedly discovered for the first time that the expression of PKMYT1 gene or its protein in pancreatic cancer tissue is significantly higher than that of PKMYT1 gene or its protein in normal tissue. The inventor also unexpectedly discovered that the overall survival of patients with high expression of PKMYT1 is significantly shortened compared with patients with low expression. Therefore, PKMYT1 gene or its protein can be used as a marker for judging the prognosis and survival of pancreatic cancer patients. In addition, the applicant also unexpectedly discovered that inhibitors of PKMYT1 gene or its protein can effectively (a) inhibit the growth and / or proliferation of pancreatic cancer cells; and / or (b) prevent and / or treat pancreatic cancer, and inhibitors of PKMYT1 gene or its protein can be used in combination with other drugs for preventing and / or treating pancreatic cancer (gemcitabine, albumin-bound (nab) paciltaxel, FOLFIRINOX combination drugs). On this basis, the inventor completed the present invention.

[0243] As used herein, the term "RP-6306" has the structural formula:

[0244]

[0245] As used herein, the term "Dasatinib" has the structural formula:

[0246]

[0247] As used herein, the structural formula of the term "PD0166285" is:

[0248]

[0249] Pancreatic cancer

[0250] Pancreatic cancer (PC) is a malignant cancer with a high probability of metastasis and high mortality. Pancreatic ductal adenocarcinoma (PDAC) is the most common type, accounting for more than 90%, so this article mainly discusses PDAC. Due to the lack of early clinical manifestations and screening and diagnostic methods, 90% of pancreatic cancers are diagnosed in the late stage when the lesions spread beyond the pancreas, and >50% of cases are accompanied by systemic metastasis. Only 11% of patients have only local lesions at the time of diagnosis, and about 52% of PDAC patients have basically no hope of treatment because of metastasis at the time of diagnosis. The most common metastatic site is the liver. At the same time, the extensive heterogeneity of gene mutations and the dense interstitial environment make PDAC one of the most chemo-suppressive cancers. The available treatment options at this stage include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, most treatments are palliative, with the aim of alleviating disease-related symptoms and prolonging life. So far, the few FDA-approved chemotherapy and targeted therapies have only increased the 5-year overall survival rate for pancreatic cancer patients from about 2% 10 years ago to 12% in 2022, with a median survival of less than 11 months (median survival is about 10-12 months, compared with 5-6 months without treatment, because many patients are diagnosed at a later stage and a lower proportion of patients are eligible for surgical resection).

[0251] sample

[0252] As used herein, the term "sample" or "specimen" refers to a material that is specifically associated with a subject, from which specific information about the subject can be determined, calculated, or inferred. A sample can be composed in whole or in part of biological material from the subject. A sample can also be a material that has been in contact with the subject in a manner that allows testing of the sample to provide information about the subject. A sample can also be a material that has been in contact with other materials that are not from the subject, but that enable the first material to be subsequently tested to determine information about the subject, such as a cleaning fluid from a probe or scalpel. A sample can be a source of biological material other than that in contact with the subject, as long as a person skilled in the art can still determine information about the subject from the sample.

[0253] Express

[0254] As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of proteins encoded by RNA or genes or gene portions, and also includes the appearance of detection substances related to expression. For example, cDNA, the binding of binding partners (such as antibodies) to genes or other oligonucleotides, proteins or protein fragments, and the chromogenic portion of the binding partners are all included within the scope of the term "expression". Therefore, the increase in half-point density on immunoblots such as western blotting is also within the scope of the term "expression" based on biological molecules.

[0255] Reference value

[0256] As used herein, the term "reference value" refers to a value that is statistically correlated with a particular result when compared to the results of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of studies comparing the expression of PKMYT1 protein with known clinical results. Some such studies are shown in the Examples section of this article. However, studies from the literature and user experience of the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by considering circumstances and results that are particularly relevant to the patient's medical history, genetics, age, and other factors.

[0257] In the present invention, the reference value refers to a cut-off value, which refers to the ratio of the expression level of PKMYT1 in pancreatic cancer tissue to the expression level of PKMYT1 in general pancreatic tissue, and preferably the relative expression level is 2 (average value).

[0258] Non-pancreatic cancer samples

[0259] As used herein, the term "non-pancreatic cancer sample" includes, but is not limited to, non-pancreatic cancer tissues of a person not suffering from pancreatic cancer and a pancreatic cancer patient.

[0260] PKMYT1 protein and polynucleotide

[0261] In the present invention, the terms "protein of the present invention", "PKMYT1 protein" and "PKMYT1 polypeptide" are used interchangeably and refer to proteins or polypeptides having the amino acid sequence of PKMYT1. They include PKMYT19 proteins with or without the initiator methionine. In addition, the term also includes full-length PKMYT1 and fragments thereof. The PKMYT1 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutants and its functionally active fragments.

[0262] PKMYT1 (protein kinase, membrane associated tyrosine / threonine 1) protein is a protein kinase encoded by the PKMYT1 gene located in band 3, subband 3, region 1 of the short arm of chromosome 16, and is a member of the WEE kinase family.

[0263] The full length of the human PKMYT1 protein is 499 amino acids (Accession No. NP_004194.3), and the full length of the mouse PKMYT1 protein is 490 amino acids (Accession No. NP_075545.2).

[0264] In the present invention, the terms "PKMYT1 gene" and "PKMYT1 polynucleotide" are used interchangeably and refer to nucleic acid sequences having a PKMYT1 nucleotide sequence.

[0265] The genomic length of the human PKMYT1 gene is 7639 bp (NCBI GenBank accession number is NC_000016), and the mRNA sequence of its transcription product is 2061 bp (NCBI GenBank accession number is NM_004203.5 ).

[0266] The full length of the mouse Pkmy1 gene genome is 10325 bp (NCBI GenBank accession number is NC_000083), and the full length of its transcription product mRNA sequence is 2040 bp (NCBI GenBank accession number is NM_023058.4).

[0267] The similarity between human and mouse PKMYT1 is 86% at the DNA level and 89% at the protein sequence level.

[0268] It should be understood that when encoding the same amino acid, the substitution of nucleotides in the codon is acceptable. It should also be understood that when conservative amino acid substitutions are produced by nucleotide substitutions, the change of nucleotides is also acceptable.

[0269] In the case of obtaining the amino acid fragment of PKMYT1, a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragment can usually be obtained by PKMYT1 amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the PKMYT1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify and obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order.

[0270] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. 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.

[0271] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0272] At present, the DNA sequence encoding the protein of the present invention (or its fragment, derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0273] The polynucleotide sequence of the present invention can be used to express or produce a recombinant PKMYT1 polypeptide by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0274] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human PKMYT1 polypeptide of the present invention, or a recombinant expression vector containing the polynucleotide;

[0275] (2) Host cells cultured in a suitable culture medium;

[0276] (3) Isolate and purify proteins from culture medium or cells.

[0277] In the present invention, the PKMYT1 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0278] Methods well known to those skilled in the art can be used to construct expression vectors containing PKMYT1 encoding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation.

[0279] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait 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 Escherichia coli.

[0280] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0281] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.

[0282] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0283] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0284] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. 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 a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, 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.

[0285] Specific antibodies

[0286] In the present invention, the terms "antibody of the present invention" and "anti-PKMYT1 specific antibody" are used interchangeably.

[0287] The present invention also includes polyclonal antibodies and monoclonal antibodies specific for human PKMYT1 polypeptides, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to the human PKMYT1 gene product or fragment. Preferably, it refers to antibodies that can bind to the human PKMYT1 gene product or fragment but do not recognize and bind to other non-related antigen molecules. The antibodies in the present invention include those molecules that can bind to and inhibit human PKMYT1 protein, and also include those antibodies that do not affect the function of human PKMYT1 protein. The present invention also includes antibodies that can bind to modified or unmodified forms of human PKMYT1 gene products.

[0288] 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., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of mouse antibodies but still retain antibody portions from humans.

[0289] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human PKMYT1 gene products or antigenic fragments thereof can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human PKMYT1 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibodies of the present invention can 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., InMonoclonalAntibodiesandTCellHyb ridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies that can block the function of human PKMYT1 protein and antibodies that do not affect the function of human PKMYT1 protein. The various antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of human PKMYT1 gene products. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of human PKMYT1 gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., 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 (e.g., yeast or insect cells).

[0290] Antibodies against human PKMYT1 protein can be used in immunohistochemistry techniques to detect human PKMYT1 protein in specimens (especially tissue samples).

[0291] Detection Methods

[0292] The present invention also provides a method for detecting pancreatic cancer by utilizing the fact that PKMYT1 exists in pancreatic cancer cells or tissues and is closely related to the risk of pancreatic cancer.

[0293] In a preferred embodiment of the present invention, the present invention provides a high-throughput second-generation sequencing method for detecting PKMYT1 as well as Sanger sequencing, fluorescence quantitative PCR (qPCR), in situ immunofluorescence (FISH) and immunohistochemistry.

[0294] Detection Kits

[0295] Based on the correlation between PKMYT1 and pancreatic cancer, that is, PKMYT1 exists in pancreatic cancer tissue, PKMYT1 can be used as a diagnostic marker for pancreatic cancer.

[0296] The present invention also provides a kit for determining the prognosis and survival period of pancreatic cancer patients, which contains a detection reagent for detecting PKMYT1 gene, mRNA, cDNA, or protein; and a label or instructions, wherein the label or instructions indicate that the kit is used to determine the prognosis and survival period of pancreatic cancer patients.

[0297] The label or instructions shall state the following:

[0298] When the ratio of the expression level of PKMYT1 E1 in the pancreatic cancer cells or tissues of the test subject to the expression level of PKMYT1 E2 in the general pancreatic cells or tissues is ≥2, it indicates that the probability of poor prognosis and shortened survival of the test subject is higher than that of the general population;

[0299] Wherein, the E2 is the expression level of PKMYT1 in general pancreatic cells or tissues of the general population.

[0300] Detection methods and kits

[0301] The present invention relates to a diagnostic test method for quantitatively and positionally detecting the level of human PKMYT1 protein or mRNA. These tests are well known in the art. The level of human PKMYT1 protein detected in the test can be used to diagnose (including auxiliary diagnosis) the prognosis and survival period of pancreatic cancer patients.

[0302] A method for detecting whether PKMYT1 protein exists in a sample is to use a specific antibody for PKMYT1 protein for detection, which includes: contacting the sample with the specific antibody for PKMYT1 protein; observing whether an antibody complex is formed, and the formation of the antibody complex indicates that PKMYT1 protein exists in the sample.

[0303] PKMYT1 protein or its polynucleotide can be used for prognosis diagnosis and treatment of PKMYT1 protein related diseases. Part or all of the polynucleotide of the present invention can be fixed on a microarray or DNA chip as a probe for differential expression analysis and gene diagnosis of genes in tissues. Anti-PKMYT1 antibodies can be fixed on a protein chip for detecting PKMYT1 protein in a sample.

[0304] The main advantages of the present invention include:

[0305] (1) The present invention first discovered that the expression of PKMYT1 gene or its protein in pancreatic cancer tissue is significantly higher than that in normal tissue. The inventors also unexpectedly discovered that the overall survival of patients with high PKMYT1 expression is significantly shortened compared with patients with low PKMYT1 expression. Therefore, PKMYT1 gene or its protein can be used as a marker for (i) detecting pancreatic cancer; and / or (ii) determining the prognosis and survival of pancreatic cancer patients. In addition, the applicants also unexpectedly discovered that inhibitors of PKMYT1 gene or its protein can effectively (a) inhibit the growth and / or proliferation of pancreatic cancer cells; and / or (b) prevent and / or treat pancreatic cancer, and that inhibitors of PKMYT1 gene or its protein can be used in combination with other drugs for preventing and / or treating pancreatic cancer (gemcitabine, albumin-bound (nab) paciltaxel, FOLFIRINOX combination drugs, or combinations thereof).

[0306] Use in combination.

[0307] (2) The present invention first discovered that p53 deficiency, dysfunction, and G1 / S cell cycle checkpoint disorders increase the sensitivity of PKMYT1 inhibitors.

[0308] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0309] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0310] General Methods

[0311] 1. Cell culture.

[0312] Pa-Tu-8988T (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ#ACC-162), MiaPaCa2 (American Type Culture Collection, ATCC#CRL-1420), KP4 (Riken#RCB1005), HPAF-II (ATCC#CRL-1997) cells and primary cells were cultured in DMEM / F12 (HyClone; #SH30023.01) medium, and YAPC (DSMZ#ACC-382), HEK293T (ATTC#ACS-4500), BxPC-3 (ATCC#CRL-1687), SNU-324 (Harvard Medical School Dr. Li Chen), Capan-1 (ATCC#HTB-79), AsPC-1 (ATCC#CRL-1682) and Panc.03.27 (ATCC#CRL-2549) cells. 1% penicillin / streptomycin (ThermoFisher Scientific; #15140122) and 10% fetal bovine serum (Thermo Fisher Scientific#10099141) were added to the culture medium to prepare a complete medium, and cultured in a cell culture incubator at 37°C with a carbon dioxide content of 5%.

[0313] 2. Library screening.

[0314] General method. See the literature Nat Protoc. 2017Apr; 12(4): 828-863. doi: 10.1038 / nprot.2017.016. Epub 2017Mar 23. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

[0315] Pa-Tu-8988T and YAPC were transduced with the GeCKOv2 library at a low infection multiplicity (MOI of approximately 0.3). After 7 days of puromycin selection (Millipore #540411), the infected cells were divided into two portions, each with a library coverage greater than 300X (approximately 37 million cells). One portion was used as a sample with a population doubling number of 0 (PD0). The other portion was maintained in culture, with a library coverage of 300X maintained at each passage until the population doubling number was 14 (PD14). Genomic DNA was extracted and purified from PD0 and PD14, and the guide sequence PCR was amplified to obtain sufficient gDNA for deep sequencing.

[0316] 3. Plasmid Construction and Lentivirus Production

[0317] The PKMYT1 expression sequence cDNA was cloned into the pCDH-CMV-MCS-EF1-Puro lentiviral expression vector (SystemBiosciences #CD510B-1). The 3XFlag sequence was added in front of the amino terminus of PKMYT1 to obtain a PKMYT1 plasmid with a Flag tag. The CRISPR-resistant PKMYT1 expression plasmid was constructed by introducing synonymous mutations using the seamless cloning kit (Beyotime #D7010FT), and the PKMYT1 N238A point mutant was constructed on this basis. The PLK1 mutant plasmid was constructed using the seamless cloning kit (Beyotime #D7010FT). Short guide RNAs (sgRNAs) targeting human PKMYT1, PLK1, TNFAIP3, BIRC3, EGR1 and mouse Trp53 and Pkmyt1 were designed using the Optimized CRISPR Design web tool (http: / / crispr.mit.edu / ). The CRISPR / Cas9 tool plasmid LentiCRISPRv2 (Addgene#52961) was linearized using restriction endonuclease BsmBI-HF (NEB#R3136V), and the sgRNA annealing product was connected to the linear vector obtained after LentiCRISPRv2 digestion to construct a lentiviral plasmid (sequence information is shown in Table 1 below). This plasmid can express sgRNA and Cas9 protein at the same time, and can knock out the target gene after being packaged into a virus to infect cells.

[0318] Table 1 Genome targeting sgRNA sequences

[0319]

[0320] Short hairpin RNAs (shRNAs) targeting human PRKDC were designed using the GPP Web Portal (https: / / portals.broadinstitute.org / gpp / public / ). PRKDC shRNA lentiviral plasmids were generated by cloning these sequences into pLKO.1 (Sigmaplasmid #SHC001) (sequence information is shown in Table 2 below). Empty vectors were used as controls. Viral packaging was performed using Lipofectamine 3000 (Invitrogen #L3000015) or polyethyleneimine (Sigma #408727). Lentiviruses were harvested 24, 36, 48, and 60 h later and frozen in equal amounts at -80 °C for infection. All cells were infected with lentivirus in supernatant containing 8 μg / ml polybrene (Sigma #107689) for 16 h and treated with puromycin (Millipore #540411) 1 day after infection.

[0321] Table 2 Genome-targeting shRNA sequences

[0322]

[0323] 4. Cell viability assay, isobologram and drug combination analysis

[0324] Cell viability was detected using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega #G7573).

[0325] After virus transduction, the screened cells were plated in a 96-well cell culture plate and an appropriate amount of cells (0.75-3×10 4 ) were cultured for 24 h. The proliferation activity was detected at 4-12 days and the cell growth curve was drawn.

[0326] When studying the effect of inhibitors on cell viability, Gemcitabin, RP-6306, PD0166285 and PRKDC inhibitors were added to 96-well plates after 24 hours of cell culture to start treating cells. Cell viability was measured every 2 to 3 days. Luminescence values ​​were detected using an ELISA reader (BioTek #H1210-018), and isobolite and compound combination index analysis was performed using CalcuSyn software, version 2 (Biosoft, Cambridge, UK).

[0327] For specific methods, please refer to the literature Chou, TCDrug Combination Studies and Their Synergy Quantification Using the Chou-Talalay Method. Cancer Research 70, 440-446, doi: 10.1158 / 0008-5472. Can-09-1947 (2010). and Gabrielsson, J., Gibbons, FD & Peletier, LAMixture dynamics: Combination therapy in oncology. Eur J Pharm Sci 88, 132-146, doi: 10.1016 / j.ejps.2016.02.020 (2016).

[0328] 5. Inhibitor Treatment

[0329] Different concentration gradients of inhibitors were used to treat cells according to their sensitivity to inhibitor treatment. When verifying the regulatory effect of PKMYT1 inhibitor RP-6306 (MCE, #HY-145817A) on PLK1, CDK1 and PRKDC, a series of drug concentrations were used for Pa-Tu-8988T (0, 0.1, 0.25, 0.75 and 2.5 μM), YAPC (0, 1, 2.5, 5 and 7.5 μM) and PDAC-CN1 (0, 0.05, 0.1, 0.25, 0.5 and 1 μM). Pa-Tu-8988T (0, 0.1, 0.25 and 0.5 μM), YAPC (0, 1, 2.5 and 5 μM) and PDAC-CN1 (0, 1, 2.5 and 5 μM) were treated with specific concentrations of RP-6306 for 24h or 48h to reflect the accumulation of γH2A.X. Western blotting was performed with Pa-Tu-8988T (0, 0.1, 0.5 and 1 μM), YAPC (0, 1, 5 and 10 μM) and PDAC-CN1 (0, 0.1, 0.5 and 1 μM) treated with Dasatinib. Cells and primary cells were treated with PD0166285 (MCE, #HY-13925; 0, 0.1, 0.5 and 1 μM) for 4 h or 12 h. For the PLK1 inhibitor onvansertib (Selleck, #S7255), cells were treated for 48 h (Pa-Tu-8988T: 0, 0.05, 0.1 and 0.5 μM; YAPC: 0, 1, 5 and 10 μM). To evaluate the efficacy of RP-6306 combined with PRKDC inhibition, three concentrations of each PRKDC inhibitor (NU7441: MCE, #HY-11006; 0, 1, and 5 μM; NU7026: MCE, #HY-15719; 0, 8, and 16 μM) and PR-6306 (Pa-Tu-8988T: 0, 0.25, and 1 μM; YAPC: 0, 2.5, and 5 μM; PDAC-CN1: 0, 1, and 5 μM) were combined.

[0330] 6. Colony formation

[0331] 6.1 Plate colony formation experiment

[0332] The cells selected by puromycin for four days after viral transduction were digested and counted, and seeded in a 6-well culture plate at a lower density (Pa-Tu-8988T: 250 cells / well; YAPC: 500 cells / well; PDAC-CN1: 500 cells / well; KC: 750 per well / well; KPC#10: 750 cells / well), and 3 mL of the corresponding complete culture medium was added to each well for culture. When the color of the culture medium turned yellow and a cell clone group visible to the naked eye was formed, the bottom of the well plate was covered with 4% paraformaldehyde (Shenggong #E672002-0500) fixative and fixed for 30 minutes, and stained with crystal violet (Shenggong #E607309-0100) for 30 minutes, and the image was collected with a scanner or camera.

[0333] 6.2 Soft agar colony formation assay

[0334] 0.6% agar (BD difco #214220) solution was covered on the bottom of the 6-well plate and allowed to stand at room temperature to solidify; the cells screened four days after viral transduction were digested and counted, and 0.35% agar solution was added to the aforementioned 6-well culture plate according to a specific number (Pa-Tu-8988T: 1X10^4 cells / well; YAPC: 1X10^4 cells / well). 100 μL of fresh culture medium was added every 3 days, and when a cell clone population visible to the naked eye was formed, 0.3 mL of 1 mg / mL MTT (Sigma–Aldrich #M5655) solution was added to each well for staining for 4 hours; images were collected using a scanner or camera.

[0335] 7.Western blotting, co-immunoprecipitation and protein profiling

[0336] Whole cell lysates of cell lines were prepared using IP buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 100 mM sodium fluoride, 2 mM sodium molybdate, 30 mM sodium pyrophosphate, 5 mM EDTA, and 2 mM sodium orthovanadate) containing protease inhibitors (10 μg / mL leupeptin, 10 μg / mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 μg / mL vanadate). Cells were then lysed for 8 hours at 4°C or disrupted with an ultrasonic cell disruptor (40% Amp, 16 s) and cleared by centrifugation at 14,000 rpm for 30 minutes at 4°C. Protein concentrations in lysates were determined using Quick Start Bradford 1×Dye Reagent (Bio-Rad #5000205).

[0337] Western Blotting was performed using standard techniques. Chemiluminescence (GE Healthcare #RPN2134) was used to detect hybridization signals, and Amersham 600 imager was used to capture signals (GE Healthcare #29083461). The primary antibodies were as follows: PKMYT1 (Cell Signaling Technology #4282S), PKMYT1 (Santa Cruz #sc-74523), CDK1 (SantaCruz #sc-54), Phospho-CDK1 (T14) (Abclonal #AP0016), Histone H3 (Cell Signaling Technology #12648S), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology #9718S), PCNA (Santa Cruz#sc-56), β-actin(Sigma#A4700), GAPDH(ABMART#M20006), PLK1(abmat#A21082), PRKDC(abmat#A1419), Phospho-PRKDC(S2056)(Abclonal#AP0 621), Flag-Tag (Merck#F-1804), TNFAIP3 (Abclonal#A19128), EGR1 (Abclonal#A23424), BIRC3 (Abclonal#A0833), Myc-Tag (Abclonal#AE070; Santa Cruz#sc-40), Panphospho-Ser / Thr(Abclonal#AP0893), Phospho-PLK1-Thr210(Abclonal#AP1025), and Phospho-PLK1–Tyr217(ABMART#TA7322). Secondary antibodies were as follows: Peroxidase-AffiniPure Goat Anti-Mouse IgG(H+L)(JACKSON#115-035-003), Peroxidase-AffiniPure Goat Anti-Rabbit IgG(H+L)(JACKSON#111-035-003). For cycloheximide CHX chase assay, cells were treated with 10 μM cycloheximide CHX(Sellck#S7418), and for protein degradation related experiments, cells were treated with 10 μM MG132(Sellck#S2619), and cells were collected at the indicated times.

[0338] For immunoprecipitation, standard techniques were used. 2.5 mg of whole cell lysate was mixed with 1 μg of anti-Flag (Merck #F1804), anti-PKMYT1 (Santa Cruz #sc-74523) or IgG (Santa Cruz #sc-2025) antibody and 20 μL of protein G-sepharose (Thermo Fisher Scientific #101242), incubated overnight, then washed with IP buffer and eluted by boiling with SDS buffer. After elution, the samples were detected by SDS-PAGE and Coomassie staining (Invitrogen #LC6025). When preparing samples for mass spectrometry analysis, they were eluted with elution buffer (8M Urea (Sangon #A600148-0500) and 100mM Tris-HCl) with shaking, pH 8.0, and analyzed by mass spectrometry. IP samples and whole cell lysates were analyzed by Western Blotting.

[0339] 8. Immunohistochemical Staining

[0340] The collected tumor tissues were fixed with formalin for 24 hours and then embedded in paraffin. After dewaxing, rehydration, and antigen retrieval, the sections were blocked with 5% goat serum (Beyotime#C0265) + 2% bovine serum albumin (Absin#abs9157) + 3% triton X-100, incubated with primary antibody (PKMYT1, Cell Signaling Technology#4282S) overnight, washed with PBS, incubated with secondary antibody (Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L), JACKSON#111-035-003) for 1 hour, washed, dried, and sealed.

[0341] 9. Cell Cycle Analysis

[0342] Propidium iodide (PI, Sigma #P4170-10MG) was used to stain the genome and analyze the cell cycle. The infected cells were synchronized for cell cycle. After recovery, the cells were digested and counted to obtain about 1×10 6 The cells were resuspended in precooled 75% ethanol and placed at -20°C for more than one day. After removing RNA, 2 μL of 5 mg / mL PI was added to every 100 μL, and mixed by pipetting. The mixture was incubated at room temperature in the dark for 10 min. Flow cytometry was used for analysis.

[0343] 10. Immunofluorescence

[0344] The infected and screened cells were digested and counted, resuspended in complete medium and inoculated in a 24-well plate, so that the cells were evenly distributed on the coverslip and cultured to a density of 50%-70%; fixed with 4% paraformaldehyde (Shenggong #E672002-0500), and left at room temperature for 10 minutes; permeabilized and blocked before incubation. Primary antibody: Phospho-Histone H2A.X (Ser139) (CellSignaling Technology #9718S); Secondary antibody: Fluorescein (FITC)-AffiniPure Goat Anti-Rabbit IgG (H+L) (JACKSON #111-095-003). 15 μL of anti-quenching sealant (Keygen Bio #KGF028) was added to the slide and the image was collected.

[0345] 11. Cell Line-Derived and Patient-Derived Xenotransplantation

[0346] Female BALB / c nude mice (5-6 weeks old; weight 18-25 g) were obtained from Shanghai Lingchang Biotechnology Co., Ltd. All animals were housed in a specific pathogen-free (SPF) facility at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, in accordance with international, national, and institutional guidelines for the humane treatment of animals and in compliance with relevant laws (SIBS-2019-WYX-1; SINH-2020-WYX-1; SINH-2021-WYX-1; SINH-2022-WYX-1). The mouse studies were approved by the Animal Care and Use Committee (IACUC) of the Shanghai Institutes for Biological Sciences. Pa-Tu-8988T (2X10^6 cells / mouse), YAPC (2X10^6 cells / mouse), PDAC-CN1 (2X10^6 cells / mouse), KC (2X10^6 cells / mouse), and KPC#10 (1.75X10^6 cells / mouse) transduced with PKMYT1 sgRNAs or Ctrl were injected into the right flank of mice. There were 6 mice in each group. Tumor xenografts grew for approximately 3-6 weeks. When the maximum tumor diameter approached the maximum tumor diameter allowed by our institutional protocol (2.0 cm) or met the observation criteria, all mice were sacrificed and tumors were collected. The maximum tumor diameter allowed by the institutional animal use committee was 2.0 cm.

[0347] For in vivo inhibitor treatment, when tumors reached a size of 100–150 mm3, mice were randomly divided into treatment and control groups (n=6 per group) based on tumor volume and body weight, and RP-6306 treatment was started. RP-6306 was prepared with 1.5% DMSO + 40% PEG300 (Selleck #S6704) + 5% Tween-80 (Selleck #S6702) + 53.5% saline and gavaged twice daily (BID, 0-8 hours) for up to 15 days. The control consisted of the above solvent. For xenografts from patients, tumor tissues were collected and cut into small pieces (approximately 2 mm in diameter). These tumor fragments were subcutaneously inoculated into the right flank of nude mice for proliferation. When the average tumor size reached approximately 150 (100-200) mm 3 At 14:00 p.m., mice were randomly divided into treatment group or solvent group (n=8 per group) according to growth rate. The tumor volume, clinical signs and body weight of the animals were monitored. The tumor volume was measured with a digital caliper and the calculation formula was: tumor volume = 0.5×L×W×W, where L is length and W is width. After the last administration, fasting whole blood was collected 8 hours later. After the administration of RP-6306, blood was collected from 6 mice in each group for routine liver function and peripheral blood tests.

[0348] 12. Liver function test

[0349] The liver function of mice after drug treatment was tested using the aspartate aminotransferase (AST) kit (Shenso Youfu #3050280) and the alanine aminotransferase (ALT) kit (Shenso Youfu #3040280). After the drug treatment, the mice were deprived of food for 8 hours; fasting blood was collected and added to the EDTA anticoagulant tube and mixed; serum was collected and liver function tested according to the instructions, and the centrifugation conditions were: 3000rpm, 15min.

[0350] 13. Routine blood test of mice

[0351] After the administration, the mice were deprived of food for 8 hours; fasting blood was collected and added to an EDTA anticoagulant tube and mixed; 20 μL of whole blood was taken and tested on a fully automatic blood analyzer.

[0352] 14. In vitro phosphorylation

[0353] Recombinant Flag-PKMYT1 protein, recombinant Myc-PLK1 (Myc-CDK1 as control) protein, ATP (Biyuntian #D7378), phosphatase inhibitor cocktail (Biyuntian #D7378), RP-6306 (MCE #HY-145817A, solvent as control) and kinase buffer (Cell Signaling Technology #9802) were incubated at 37°C for 1 hour, and then 2.5× protein loading buffer was added to stop the reaction on ice and boiled at 95°C for 5 minutes. Phosphorylation detection was performed by Western Blotting experiment.

[0354] Example 1 Whole-genome CRISPR (clustered regularly interspaced short palindromic repeats) screening identifies PKMYT1 as a weak spot in pancreatic ductal adenocarcinoma (PDAC)

[0355] Experimental steps:

[0356] 1) Perform genome-wide CRISPR knockout screening in two pancreatic cancer cell lines (YAPC and Pa-Tu-8988T) to identify genes that inhibit cell proliferation after knockout;

[0357] 2) Comprehensively analyze the aforementioned CRISPR screening data, cell line transcriptome sequencing data, CRISPR screening data in other gastrointestinal stromal tumors (GIST), and reported druggable genome information, eliminate essential genes, screen key genes that affect PDAC proliferation, and find the weaknesses of PDAC.

[0358] Experimental results:

[0359] The results are as follows Figure 1 shown.

[0360] Figure 1 Shown is the identification of the PDAC-dependent gene PKMYT1 by a genome-wide CRISPR loss-of-function screen.

[0361] CRISPR Screening Figure 1 a), by comparing the sequencing data, it was found that in the PD14 sample, the cumulative frequency curve of sgRNA was shifted and the abundance distribution was wider, indicating that cells with proliferation advantages were enriched during the experiment, while sgRNAs that were not conducive to cell proliferation were gradually eliminated, further indicating that screening was achieved during the entire experiment ( Figure 1 b). There is a significant correlation between the A and B libraries of hGeCKOv2 and between the cell lines, indicating that the repeatability and reproducibility of the screening are very high ( Figure 1cd). The med.CS and β-score of genes were calculated using different algorithms, and there was also a significant correlation between the two, indicating that the data analysis method was reasonable ( Figure 1 e). Functional gene set analysis found that genes involved in a variety of basic biological activities of cells were in the top 1% of negative screening ( Figure 1 g). This indicates that the CRISPR / Cas9 screening system and analysis method are effective and feasible ( Figure 1 ag).

[0362] By comparing the PDAC cell line screening data with the gastrointestinal stromal tumor screening data ( Figure 1 h), a list of candidate target genes specific for pancreatic cancer was obtained, and then combined with the known core essential genes, non-essential genes, druggable genome, and the correlation between gene expression and prognosis ( Figure 2 d) and locked in the PKMYT1 gene as a candidate gene screened by CRISPR / Cas9.

[0363] The results showed that CRISPR / Cas9 screening technology can quantify gene-specific phenotypic changes. Combining multiple data analyses, the PKMYT1 gene was identified as a candidate gene for CRISPR / Cas9 screening.

[0364] Example 2 PKMYT1 is highly expressed in PDAC, and high PKMYT1 expression levels are associated with malignant progression and poor prognosis of tumors

[0365] Experimental steps:

[0366] 1) Immunohistochemical staining was performed on samples from two clinical patients to detect the expression level of PKMYT1;

[0367] 2) Analyze the relationship between PKMYT1 expression levels and tumor progression and differentiation in PDAC patient samples in public databases;

[0368] 3) Analyze the PKMYT1 expression levels in PDAC patient samples and corresponding normal pancreatic tissues in public databases;

[0369] 4) Analyze the amplification of the PKMYT1 gene in PDAC patient samples in public databases;

[0370] 5) Analyze the relationship between PKMYT1 expression level and patient prognosis in multiple data sets.

[0371] Experimental results:

[0372] The results are as follows Figure 2 shown.

[0373] Figure 2 It shows that PKMYT1 is highly expressed in PDAC and has significant clinical significance. Analysis of the expression data of PDAC clinical samples found that the expression of PKMYT1 gene in cancer tissues was higher than that in normal samples (the ratio of the mean was significantly greater than 2) ( Figure 2 c), and the expression level is positively correlated with the degree of tumor differentiation and malignancy ( Figure 2 b). There is a certain degree of genomic amplification and high expression in tumor samples ( Figure 2 a, 2e). Combined with the patient's clinical data, it was found that high expression of PKMYT1 was associated with poor prognosis and shortened the patient's overall survival ( Figure 2 d).

[0374] Example 3 PKMYT1 gene deletion inhibits the proliferation activity of PDAC in vivo and in vitro, induces G2 / M arrest of the cell cycle, accumulates a large amount of pan-nuclear DNA damage, and produces an apoptotic phenotype

[0375] Experimental steps:

[0376] 1) Construction of PKMYT1 gene lentiviral knockout plasmids (CRISPR / Cas9 technology, plasmids PKMYT1-sg2 and PKMYT1-sg3);

[0377] 2) After packaging, the lentivirus was used to infect Pa-Tu-8988T, YAPC, KP-4, MiaPaCa-2, BxPC-3 and SNU324 cells as well as PDAC primary cultured cells PDAC-CN1 and PDAC-CN2;

[0378] 3) Immunoblotting to detect PKMYT1 knockout levels;

[0379] 4) Extract RNA for transcript sequencing;

[0380] 5) Detect cell viability using the CellTiter-Glo (CTG) kit;

[0381] 6) Plate colony formation assay and soft agar colony formation assay were used to detect the effect of PKMYT1 knockout on cell proliferation;

[0382] 7) PKMYT1 knockout Pa-Tu-8988T, YAPC and PDAC-CN1 cells and the control group were injected subcutaneously into nude mice to establish xenograft tumors, and the tumor growth was regularly observed and detected;

[0383] 8) Flow cytometry was used to detect the cell cycle distribution of each cell after PKMYT1 knockout;

[0384] 9) Immunoblotting and immunofluorescence methods were used to detect the degree of DNA damage;

[0385] 10) Flow cytometry was used to detect the apoptosis of each cell after PKMYT1 knockout.

[0386] 11) Pa-Tu-8988T and YAPC with PKMYT1 knockout were treated with gemcitabine, and cell viability was detected.

[0387] Experimental results:

[0388] The results are as follows Figure 3 and 4 shown.

[0389] Figure 3 We showed that PKMYT1 knockout inhibited the growth and proliferation of PDAC in vivo cell models and in vitro xenograft models.

[0390] Figure 4 It was shown that PKMYT1 knockout inhibited the growth and proliferation of PDAC in vitro.

[0391] Cell viability assays, plate anchorage-dependent and soft agar anchorage-independent colony formation assays all showed that after knocking out PKMYT1 under in vitro culture conditions, the proliferation activity of PDAC tumor cells weakened ( Figure 3 ad, Figure 4 ab). Mouse subcutaneous xenograft tumor experiments also showed that PKMYT1 is essential for PDAC proliferation under in vivo conditions ( Figure 3 Flow cytometry analysis of apoptosis revealed that knockout of PKMYT1 in PDAC significantly increased the level of cell apoptosis, and transcriptome analysis showed that apoptosis-related signaling pathways were also enriched after PKMYT1 was targeted ( Figure 3 j, Figure 4 ef), the expression of PCNA protein was not regulated in Western Blotting analysis ( Figure 4 c), indicating that the function of PKMYT1 affects the proliferation activity and rate of PDCA mainly by regulating cell apoptosis rather than by changing the cell proliferation ability. Western Blotting experiments showed that the knockout of PKMYT1 reduced the phosphorylation of threonine 14 of CDK1 ( Figure 4 c). Transcriptome differential expression analysis showed that the gene sets related to mitotic spindle formation and G2 / M cycle checkpoint were enriched after PKMYT1 knockout ( Figure 4 f). Flow cytometry analysis showed that PKMYT1 knockout altered cell cycle progression, causing G2 / M cycle arrest and ultimately inhibiting PDAC proliferation ( Figure 3 h, Figure 4d). In Western Blotting and immunofluorescence experiments, PKMYT1 gene editing caused phosphorylation of H2A.X in cells, resulting in pan-nuclear DNA damage ( Figure 3 i, Figure 4 c). This indicates that kinase deficiency interferes with the G2 / M checkpoint, prompting cells to enter mitosis prematurely, accumulating a large amount of DNA damage, leading to genomic instability, accumulating more and more DNA damage, and then producing mitotic catastrophes, and finally stagnating in the G2 / M phase. Knockout of PKMYT1 increases the sensitivity of PDAC to gemcitabine ( Figure 4 g).

[0392] Example 4 PKMYT1 inhibitor RP-6306 can inhibit the proliferation of PDAC in vitro, induce G2 / M arrest of the cell cycle, accumulate a large amount of pan-nuclear DNA damage, and produce an apoptotic phenotype; at the same time, it can effectively inhibit the in vivo proliferation of PDAC cell line-derived xenograft tumor models (CDX) and patient-derived xenograft tumor models (PDX), and will not produce obvious toxic side effects. Other broad-spectrum kinase inhibitors that can target PKMYT1, such as Dasatinib and PD0166285, can also inhibit the proliferation of pancreatic cancer and produce related phenotypes such as cycle arrest, DNA damage, and apoptosis.

[0393] Experimental steps:

[0394] 1) The effect of RP-6306 on cell proliferation activity was detected in 4 PDAC cell lines and 2 primary cultured cell lines, and its half effective concentration (IC 50 );

[0395] 2) Treat Pa-Tu-8988T, YAPC and PDAC-CN1 cells with concentration gradient of inhibitor RP-6306;

[0396] 3) Immunoblotting to detect the phosphorylation levels of PKMYT1 and CDK1;

[0397] 4) Detect cell viability using CTG kit;

[0398] 5) Extract RNA for transcript sequencing;

[0399] 6) Flow cytometry was used to detect the cell cycle distribution of each cell after inhibitor treatment;

[0400] 7) Immunoblotting method to detect the degree of DNA damage;

[0401] 8) Detect cell apoptosis by flow cytometry;

[0402] 9) Pa-Tu-8988T cells were injected subcutaneously into nude mice to establish xenograft tumors. RP-6306 was used to detect the effect on the tumor size and volume of CDX. At the same time, liver function tests and blood routine tests were performed to study whether RP-6306 would have toxic effects on mice;

[0403] 10) Construct patient-derived PDX and use RP-6306 to detect the effects on tumor size and volume of PDX. At the same time, use liver function tests and blood routine tests to study whether RP-6306 will have toxic effects on mice.

[0404] 11) Treat pancreatic cancer cells with other PKMYT1 inhibitors, such as Dasatinib and PD0166285, and perform relevant analyses such as cell viability, immunoblotting, and flow cytometry.

[0405] Experimental results:

[0406] The results are as follows Figure 5-9 shown.

[0407] Figure 5 The PKMYT1 inhibitor RP-6306 was shown to inhibit tumor growth and proliferation in multiple PDAC cell models in vitro.

[0408] Figure 6 The PKMYT1 inhibitor RP-6306 was shown to inhibit the in vivo growth of cell line-derived xenografts as well as xenografts derived from tumor patients without producing significant toxic side effects.

[0409] Figure 7 It was shown that the PKMYT1 inhibitor RP-6306 can inhibit the in vivo growth and proliferation of cell-derived xenografts and xenografts derived from tumor patients without producing obvious toxic side effects.

[0410] Figure 8 We show that the PKMYT1 inhibitor PD0166285 can suppress proliferation in multiple PDAC models and produce apoptotic and cycle arrest phenotypes consistent with PKMYT1 inhibition.

[0411] Fig. 9 The PKMYT1 inhibitor Dasatinib was shown to inhibit proliferation of multiple PDAC models and produce apoptosis and cell cycle arrest phenotypes consistent with PKMYT1 inhibition. Treatment with the PKMYT1-specific inhibitor RP-6306 effectively inhibited the in vitro proliferation of pancreatic cancer cell lines and primary cultured cell lines ( Figure 5 a). RP-6306 inhibitor treatment increased the hyperphosphorylation level of PKMYT1 in a drug concentration-dependent manner ( Figure 5b), inhibiting its classical CDK1 kinase activity and downregulating the phosphorylation of the Thr14 site of CDK1 ( Figure 5 b), accompanied by an increase in massive cellular DNA damage ( Figure 5 c) As the drug concentration increases, the cell cycle arrest and apoptosis degree also increase gradually ( Figure 5 de). In mouse subcutaneous cell line xenograft tumor experiments and PDX models, RP-6306 did not produce obvious toxic side effects (such as mouse body weight, liver function damage, blood routine, etc.) Figure 6 c,6f,6g-h, Figure 7 ab), can effectively slow down the tumor growth rate of tumor cell lines and patient tissues in mice, and even achieve tumor regression, achieving a tumor suppressor effect ( Figure 6 ab,6d-e).

[0412] Inhibitors Dasatinib and PD0166285 inhibit PDAC cell proliferation ( Figure 8 b, Fig. 9 b), induces accumulation of DNA damage and cell cycle arrest ( Figure 8 c,8e, Fig. 9 d), and at the same time, cells showed significant apoptosis ( Figure 8 d, Fig. 9 c). Both inhibitors are broad-spectrum inhibitors, and PKMYT1 is not their main target, but treatment with the compound can indeed regulate the kinase activity of PKMYT1 ( Figure 8 a, Fig. 9 a), affecting the phosphorylation level of CDK1 in cells ( Figure 8 a, Fig. 9 a), indicating that the inhibitor exerts its effect in PDAC to a certain extent by regulating the function of PKMYT1.

[0413] Example 5 In PDAC, PKMYT1 can regulate multiple signaling pathways. PKMYT1 interacts with PLK1 and can exert oncogene function by regulating the expression of PLK1.

[0414] Experimental steps:

[0415] 1) We constructed an sgRNA-tolerant PKMYT1 overexpression lentiviral plasmid (PKMYT1-sgR), and based on it, mutated asparagine at position 238 to alanine (N238A, PKMYT1-NA), which would cause PKMYT1 to lose its kinase activity. We combined PKMYT1 knockout with PKMYT1-sgR or PKMYT1-NA to study the effect of PKMYT1's classical kinase activity on proliferation;

[0416] 2) Construction of Flag-PKMYT1 exogenous expression plasmid;

[0417] 3) Flag-PKMYT1 was overexpressed in Pa-Tu-8988T, and PKMYT1-interacting proteins were pulled down using Flag antibody and identified by mass spectrometry;

[0418] 4) Construct Flag-PLK1 exogenous expression plasmid and verify the relationship between PKMYT1, PLK1 and PRKDC in Pa-Tu-8988T and YAPC cells.

[0419] 5) Knock out PKMYT1 in Pa-Tu-8988T, YAPC and PDAC-CN1 cells or use the inhibitor RP-6306 with a concentration gradient, or construct an exogenous expression plasmid of PKMYT1 and overexpress it in the cell model, and use immunoblotting to detect the regulation of PLK1 after PKMYT1 inhibition or overexpression;

[0420] 6) Treat PKMYT1 knockout cells with cycloheximide (CHX) or MG132, and use immunoblotting to detect the regulation of PKMYT1 deficiency on PLK1 expression;

[0421] 7) Construction of PLK1 exogenous expression plasmid and PLK1 gene lentiviral knockout plasmid (CRISPR / Cas9 technology, plasmids PLK1-sg1 and sg2);

[0422] 8) After knocking out PKMYT1 in YAPC (CRISPR / Cas9 technology), wild-type PLK1, PLK1 with tyrosine 217 mutated to aspartic acid (PLK1 Y217D) or phenylalanine (PLK1 Y217F) were overexpressed, and the restoration of PDAC proliferation activity was detected by CTG experiment;

[0423] 9) Package lentivirus and infect three PDAC cell lines to verify whether PLK1 deficiency can reproduce the effects of PKMYT1 inhibition on cell cycle, DNA damage and apoptosis;

[0424] 10) In vitro kinase assays were used to verify the phosphorylation of PLK1 by PKMYT1.

[0425] Experimental results:

[0426] The results are as follows Figure 10-11 shown.

[0427] Fig.10 We show that PKMYT1 interacts with PLK1 and PRKDC in PDAC and that PLK1 mediates the oncogenic role of PKMYT1 in pancreatic cancer.

[0428] Fig.11 It was shown that in PDAC, PKMYT1 can regulate PLK1 expression in addition to its classical kinase function.

[0429] Under the condition of PKMYT1 knockout, after complementing the wild-type PKMYT1 protein tolerated by sgRNA, the cell proliferation rate was observed to be restored to a certain extent by the cell viability detection experiment, while complementing the N238A mutant PKMYT1 protein could not improve the cell proliferation activity. The above results indicate that the classical functional kinase activity of PKMYT1 protein is at least partially involved in the regulation of PDAC tumor proliferation ( Fig.11 ab).

[0430] Immunoprecipitation and protein mass spectrometry analysis revealed that PKMYT1 interacts with PLK1 in PDAC ( Fig.10 ab, Fig.11 c). Immunoblotting experiments showed that knockout of PKMYT1 or treatment with a specific inhibitor would downregulate the protein expression level of PLK1, while exogenous overexpression of PKMYT1 would increase the expression level of PLK1 ( Fig.10 ce). Combined with cycloheximide treatment, we demonstrate that inhibition of PKMYT1 shortens the lifespan of PLK1 protein, resulting in a shorter half-life of the protein ( Fig.10 f). Under MG132 treatment, we demonstrated that PKMYT1 knockout modulates the proteasome-dependent degradation of PLK1 ( Fig.10 g). At the same time, analysis of pancreatic cancer clinical patient sample transcription and prognosis data found that the expression levels of PKMYT1 and PLK1 were strongly correlated, and high expression of PLK1 was also associated with poor prognosis ( Fig.11 In addition, functional inhibition of PLK1 can reproduce the various phenotypes of PKMYT1 inhibition in PDAC, in addition to DNA damage accumulation, such as cell proliferation inhibition, cell cycle arrest, and apoptosis ( Fig.10 hj, Fig.11 fg), overexpression of PLK1 alleviates the PDAC proliferation inhibition caused by PKMYT1 knockout ( Fig.10 k), indicating that PKMYT1 can regulate PDAC by regulating PLK1. PKMYT1 can phosphorylate the tyrosine at site 217 of the PLK1 protein and increase the phosphorylation levels of multiple serine / threonine sites ( Fig.11 h), and the phosphorylation of tyrosine at position 217 of PLK1 can improve the proliferation inhibition caused by PKMYT1 knockout to a certain extent ( Fig.11 ij).

[0431] Example 6 Inhibition of PKMYT1 by inhibitors (such as RP-6306) or genetic knockout of PKMYT1 increases the expression of ubiquitin ligases TNFAIP3 and BIRC3, as well as the transcription factor EGR1. TNFAIP3 and BIRC3 can mediate the regulation of PKMYT1 on PLK1 expression

[0432] Experimental steps:

[0433] 1) After RP-6306 treatment or genetic knockout of PKMYT1 (CRISPR / Cas9 technology, plasmid PKMYT1-sg3), RNA was extracted for transcript sequencing;

[0434] 2) Construct TNFAIP3, BIRC3 and EGR1 gene lentiviral knockout plasmids (CRISPR / Cas9 technology, plasmids sgTNFAIP3-2, sgTNFAIP3-3, sgBIRC3-2, sgEGR1-2 and sgEGR1-3), package the lentivirus and infect Pa-Tu-8988T and YAPC cells;

[0435] 3) Western blotting to verify the effect of PKMYT1 inhibition on TNFAIP3, BIRC3 and EGR1 proteins;

[0436] 4) Immunoblotting verified that TNFAIP3 and BIRC3 mediated the regulation of PKMYT1 on PLK1 expression;

[0437] 5) Overexpress the ubiquitination system in HEK293T cells to verify the ubiquitination of PLK1 by TNFAIP3.

[0438] Experimental results:

[0439] The results are as follows Fig.12 shown.

[0440] PKMYT1 knockout ( Fig.12 a, 12c) or specific inhibitor treatment ( Fig.12 b, 12d) induced increased expression of TNFAIP3, BIRC3, and EGR1, and there was a clear gradient effect when the inhibitor was treated. Among them, TNFAIP3 and BIRC3 have ubiquitin ligase function, and EGR1 is predicted to be a transcription factor of TNFAIP3. Knockout of TNFAIP3 and BIRC3 attenuated the downregulation of PLK1 caused by PKMYT1 inhibition ( Fig.12 e). TNFAIP3 can increase the ubiquitination level of PLK1 ( Fig.12 f).

[0441] Example 7 PRKDC is required for PKMYT1 inhibition to play a tumor suppressor role in PDAC. PKMYT1 inhibition and p53 inactivation in PDAC are synthetically lethal, and TP53 dysfunction increases PDAC's dependence on PKMYT1

[0442] Experimental steps:

[0443] 1) Knockout of PKMYT1 (CRISPR / Cas9 technology) or treatment with gradient concentrations of RP-6306 in Pa-Tu-8988T, YAPC, and PDAC-CN1 cells, and immunoblotting to detect the regulation of PRKDC and phosphorylated PRKDC after PKMYT1 inhibition;

[0444] 2) Knock down PRKDC (this gene has a certain degree of genomic amplification, and RNAi technology is used for interference, plasmid PRKDC-sh3) to verify whether PRKDC function affects the sensitivity of PDAC to RP-6306;

[0445] 3) The PRKDC inhibitors NU7441 or NU7026 were combined with RP-6306 at different concentrations to treat three pancreatic cancer cell lines, and the changes in γH2A.X were detected by immunoblotting. At the same time, the relationship between the inhibitor combinations (whether there is synergistic or antagonistic effect) was analyzed using the CTG experiment; 4) The relationship between the expression level of PKMYT1 in PDAC patient samples in the public database and TP53 mutation and p53 signaling pathway was analyzed;

[0446] 5) Construction of paired heteroallelic cell line models of Trp53 knockout (CRISPR / Cas9 technology);

[0447] 6) Construction of PKMYT1 (Pkmyt1) gene lentiviral knockout plasmids (CRISPR / Cas9 technology, plasmids PKMYT1-sg2, PKMYT1-sg3, Pkmyt1-msg5 and Pkmyt1-msg6);

[0448] 7) Packaged lentivirus and infected 6 pancreatic cancer cell lines;

[0449] 8) Detect cell viability using CTG kit;

[0450] 9) Plate clone formation assay to detect the effect of TP53 (Trp53) knockout on the proliferation of different cells;

[0451] 10) Knockout Trp53 in paired heteroallelic cell lines, inject them subcutaneously into nude mice to establish xenograft tumors, and regularly observe and detect tumor growth;

[0452] 11) Paired heteroallelic cell lines were treated with RP-6306, and cell viability was detected using the CTG kit.

[0453] Experimental results:

[0454] The results are as follows Fig.10 ad, as shown in 13-14.

[0455] Fig.13 It was shown that PKMYT1 inhibition in PDAC increased PRKDC phosphorylation and activation, and that inhibition of PKMYT1-induced tumor suppressor effects depended on the function of RKDC.

[0456] Fig.14 We show that TP53 mutation status influences PDAC's dependence on PKMYT1.

[0457] Immunoprecipitation and protein mass spectrometry analysis revealed that PRKDC strongly interacts with PKMYT1 in PDAC ( Fig.10 ab, Fig.13 a). Immunoblotting experiments showed that CRISPR / Cas9 editing and inhibitor treatment of PKMYT1 in PDAC increased the phosphorylation of serine 2056 of PRKDC protein and induced the activation of PRKDC protein ( Fig.10 cd). Treatment with PRKDC inhibitor significantly increased the accumulation of γH2A.X produced by PKMYT1 inhibitor, indicating that PRKDC is involved in the DNA damage response induced by PKMYT1. Fig.13 bc). RNA interference with PRKDC or PRKDC inhibitors (preventing PRKDC activation) can reduce the sensitivity of PDAC cells to PKMYT1 inhibitors, and there is a clear antagonistic effect between the inhibitors ( Fig.13 df), thus targeting PKMYT1 in PDAC to generate tumor suppressor effects requires activation of PRKDC.

[0458] Analysis of PDAC clinical sample data found that the expression of PKMYT1 was significantly correlated with the p53 signaling pathway, and the expression of PKMYT1 was higher in samples of patients with TP53 mutations ( Fig.14 ab). By constructing a paired cell model of Trp53 wild-type and mutant, we explored whether the function of p53 would affect the dependence of pancreatic cancer cells on PKMYT1 ( Fig.14 c). Short-term cell viability assays and long-term clone formation experiments showed that Trp53 knockout increased the dependence of PDAC cells on Pkmyt1 ( Fig.14 de, 14g). Compared with other TP53 mutant cell lines, TP53 wild-type normal human fibroblasts BJ and GIST430 cells are less sensitive to PKMYT1 knockout ( Fig.14 de). PKMYT1 inhibitors are more sensitive in Trp53-knockout KPC cells ( Fig.14 f). Tumor formation experiments in nude mice showed that loss of Trp53 function would increase the effect of the inhibitor ( Fig.14 h). In summary, PDAC dependence on PKMYT1 is related to the p53 signaling pathway. Dysfunction of the p53 signaling pathway increases the dependence of pancreatic cancer on PKMYT1.

[0459] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a PKMYT1 gene, mRNA, cDNA, or protein or a detection reagent thereof, characterized in that: (i) used as a marker for determining the prognosis and survival period of pancreatic cancer patients, and / or (ii) used for preparing a diagnostic reagent or kit for determining the prognosis and survival period of pancreatic cancer patients.

2. A diagnostic kit for determining the prognosis and survival period of pancreatic cancer patients, characterized in that: The kit comprises a container containing a detection reagent for detecting PKMYT1 gene, mRNA, cDNA, or protein; and a label or instruction manual, wherein the label or instruction manual indicates that the kit is used to determine the prognosis and survival period of pancreatic cancer patients.

3. A method for determining the prognosis and survival period of a pancreatic cancer patient, characterized in that: The method comprises: a) providing a test sample from a subject; b) detecting the expression level E1 of PKMYT1 protein in the test sample; and c) comparing the expression level E1 of the PKMYT1 protein determined in step b) with that of the control, Wherein, compared with the control, the expression level E1 of the PKMYT1 protein in the sample is higher than the reference value, indicating that the probability of poor prognosis and shortened survival of the subject is higher than that of the general population (control group); and / or Compared with the control, the expression level of PKMYT1 protein in the sample was lower than the reference value, indicating that The subjects had a lower chance of poor prognosis and shortened survival than the general population (control group).

4. A method for determining a treatment regimen, characterized in that: include: a) providing a test sample from a subject; b) detecting the expression level of PKMYT1 protein in the test sample; and c) determining a treatment regimen based on the expression level of PKMYT1 protein in the sample.

5. A use of a PKMYT1 gene or protein inhibitor thereof, characterized in that: Used for preparing a composition or preparation, wherein the composition or preparation is used for (a) inhibiting the growth and / or proliferation of pancreatic cancer cells; and / or (b) preventing and / or treating pancreatic cancer.

6. A pharmaceutical composition, characterized in that include: (a1) inhibitors of PKMYT1 gene or its protein; and (b) a pharmaceutically acceptable carrier.

7. A medicine box, characterized in that: include: (a1) a first container, and an inhibitor of PKMYT1 gene or protein thereof, or a drug containing an inhibitor of PKMYT1 gene or protein thereof, located in the first container.

8. Use of the pharmaceutical composition according to claim 6 or the medicine kit according to claim 7, characterized in that: For use in (a) inhibiting the growth and / or proliferation of pancreatic cancer cells; and / or (b) preventing and / or treating pancreatic cancer.

9. A non-therapeutic in vitro method for inhibiting the growth or proliferation of pancreatic cancer cells, characterized in that: The method comprises the steps of: culturing pancreatic cancer cells in the presence of a PKMYT1 gene or a protein inhibitor thereof, thereby inhibiting the growth or proliferation of pancreatic cancer cells.

10. A method for screening candidate compounds for preventing and / or treating pancreatic cancer, characterized in that: The method comprises the steps of: (a) In a test group, a test compound is added to the cell culture system, and the expression level (E1) and / or activity (A1) of PKMYT1 in the cells of the test group is observed; in a control group, no test compound is added to the same cell culture system, and the expression level (E0) and / or activity (A0) of PKMYT1 in the cells of the control group is observed; Among them, if the expression level (E1) and / or activity (A1) of PKMYT1 in the cells of the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate compound for preventing and / or treating pancreatic cancer that has an inhibitory effect on the expression and / or activity of PKMYT1.

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