WO2023149608A1 - Biomarqueur trim51 pour prédire la résistance au traitement contre le mélanome et son utilisation - Google Patents

Biomarqueur trim51 pour prédire la résistance au traitement contre le mélanome et son utilisation Download PDF

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WO2023149608A1
WO2023149608A1 PCT/KR2022/011017 KR2022011017W WO2023149608A1 WO 2023149608 A1 WO2023149608 A1 WO 2023149608A1 KR 2022011017 W KR2022011017 W KR 2022011017W WO 2023149608 A1 WO2023149608 A1 WO 2023149608A1
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melanoma
trim51
protein
mrna
resistance
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PCT/KR2022/011017
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Korean (ko)
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임병호
최길돈
조경진
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한국화학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • TRIM51 Tripartite Motif-Containing 51 mRNA or protein-containing melanoma drug resistance predictive biomarker
  • TRIM51 Tripartite Motif-Containing 51 mRNA or protein expression level measurement agent containing melanoma drug resistance as an active ingredient It relates to a composition for prediction, a kit for predicting melanoma drug resistance including the same, a method for providing information necessary for predicting resistance of a melanoma drug drug, and a method for screening a melanoma drug response enhancer.
  • Malignant melanoma refers to a malignant neoplasm (cancer) of the skin composed of melanin-producing cells. Specifically, melanin-producing cells are congenital or acquired, and melanocytes are normal cells in the skin or mucous membranes, and skin color appears due to a pigment called melanin produced by these cells. At this time, sunbathing or excessive sunlight exposure produces a lot of melanin in melanocytes and changes the skin color to black.
  • malignant melanoma Cancer arising from melanocytes that normally exist in this way is called malignant melanoma. Because melanocytes are congenitally large, they tend to develop into malignant melanoma. Smaller melanomas may be more common in fair-skinned people.
  • the malignant melanoma mainly occurs in the skin, but may also occur in other parts with mucous membranes such as the eye (eye), rectum, nose or esophagus.
  • Malignant tumors occurring in the skin include squamous cell carcinoma, basal cell carcinoma, and the like in addition to malignant melanoma, and among them, malignant melanoma is known to be the most malignant.
  • BRAF inhibitors or MEK inhibitors are recommended for the treatment of melanoma, but resistance is shown in melanoma patients. Therefore, in order to improve the cure rate of treatment for patients with advanced melanoma, it was urgently needed to establish a strategy to overcome melanoma resistance based on the interaction analysis of melanoma cells and to discover therapeutic predictive response markers for melanoma drugs.
  • An object of the present invention is to provide a biomarker for predicting melanoma treatment resistance including TRIM51 (Tripartite Motif-Containing 51) mRNA or protein.
  • TRIM51 Tripartite Motif-Containing 51
  • a composition for melanoma drug resistance prediction comprising an agent measuring the mRNA or protein expression level as an active ingredient, and a melanoma drug resistance prediction kit comprising the same do.
  • an object of the present invention is to provide a method for screening a melanoma therapeutic agent responsiveness enhancer comprising the step of selecting a test substance having a reduced mRNA or protein level of TRIM51.
  • the melanoma therapeutic agent is characterized in that it includes at least one inhibitor selected from the group consisting of a BRAF inhibitor and a MEK inhibitor.
  • the biomarker according to one aspect is a biomarker for predicting resistance to a melanoma treatment including TRIM51 (Tripartite Motif-Containing 51) mRNA or protein, wherein the melanoma treatment is one or more inhibitors selected from the group consisting of a BRAF inhibitor and a MEK inhibitor It is characterized in that it includes.
  • TRIM51 Tripartite Motif-Containing 51
  • the melanoma may be a BRAF mutant melanoma.
  • the melanoma may be at least one melanoma selected from the group consisting of cutaneous melanoma and uveal melanoma.
  • the resistance may be intrinsic resistance.
  • a composition according to another aspect is a composition for predicting resistance to a melanoma treatment comprising, as an active ingredient, an agent for measuring TRIM51 (Tripartite Motif-Containing 51) mRNA or protein expression level, wherein the melanoma treatment is a BRAF inhibitor and a MEK inhibitor. It includes one or more inhibitors selected from the group consisting of
  • the agent for measuring the mRNA expression level is a primer or probe that specifically binds to TRIM51 mRNA, and the agent for measuring the expression level of the protein may be an antibody that specifically binds to the TRIM51 protein, a fragment thereof, or an aptamer. .
  • a kit for predicting melanoma drug resistance includes the composition for predicting melanoma drug resistance.
  • the kit for predicting drug resistance to melanoma may be an RT-PCR kit, a DNA chip kit, or a protein chip kit.
  • a method for providing information includes measuring the level of mRNA or protein of TRIM51 in a sample isolated from a melanoma patient; Comparing the measured mRNA or protein level of TRIM51 with the level of TRIM51 mRNA or protein in a normal control sample; And determining that resistance to the melanoma treatment is low when the measured mRNA or protein level of TRIM51 is higher than the level of the normal control sample;
  • the melanoma treatment agent is characterized in that it includes at least one inhibitor selected from the group consisting of a BRAF inhibitor and a MEK inhibitor.
  • the step of measuring the mRNA level of TRIM51 may use a primer or probe that specifically binds to the TRIM51 mRNA.
  • the step of measuring the TRIM51 protein level may use an antibody, a fragment thereof, or an aptamer that specifically binds to the protein.
  • a screening method includes contacting a sample isolated from a melanoma patient with a test substance; Measuring the mRNA or protein level of TRIM51 in a sample contacted with the test substance; And compared to a control sample, a screening method for a melanoma treatment agent responsiveness enhancer comprising the step of selecting a test substance having a reduced mRNA or protein level of TRIM51, wherein the melanoma treatment agent is selected from the group consisting of a BRAF inhibitor and a MEK inhibitor Characterized in that it contains one or more inhibitors.
  • TRIM51 Tripartite Motif-Containing 51 as a target substance and a predictive biomarker for treatment response for overcoming resistance to melanoma therapeutics in relation to resistance to melanoma therapeutics including at least one selected from the group consisting of BRAF inhibitors and MEK inhibitors mRNA or protein was discovered.
  • melanoma-derived TRIM51 is a biomarker for predicting melanoma treatment response It is highly likely that it will be useful.
  • Figure 1a shows the genes aligned with the drug response score (DRS) converted to DRS (0.3) by calculating the pharmacologic correlation of each gene for 481 drugs of Cancer Therapeutics Response Portal (CTRP) data according to one embodiment. is the graph shown.
  • DRS drug response score
  • CRP Cancer Therapeutics Response Portal
  • Figure 1b is a graph showing the relative display of DRS for all drugs, kinase inhibitors and BRAF inhibitors according to one embodiment.
  • Figure 2 is a graph showing the TRIM51 mRNA level test using cBioPortal in various cancer cell lines (CCL).
  • FIG. 3A is a heat map of drug sensitivity profiles for several BRAF-MEK inhibitors as measured by area under the drug response curve (AUC) according to one embodiment
  • FIG. Figure 3c is a diagram showing the expression level of BRAF-MEK pathway-related proteins measured by reverse-phase protein array (RPPA) displayed in 201 cancer cell lines (CCL) sorted by TRIM51 mRNA level according to one embodiment. is the diagram shown. (At this time, parentheses indicate TRIM51 mRNA level and Pearson's correlation coefficient)
  • RPPA reverse-phase protein array
  • FIG. 4A and 4B show the effect of TRIM51 in a BRAF mutant melanoma cancer cell line (CCL) using Cancer Therapeutics Response Portal (CTRP) data (FIG. 4A) and simultaneous relative inhibition profiling (PRISM) data in mixtures (FIG. 4B).
  • CRP Cancer Therapeutics Response Portal
  • PRISM simultaneous relative inhibition profiling
  • Figure 5a is a bar graph showing the expression level of TRIM51 when A375 melanoma cells were treated with the BRAF inhibitor vemurafenib according to one embodiment.
  • Figure 5b is a bar graph showing the expression level of TRIM51 when 92.1 uveal melanoma cells were treated with the MEK inhibitor, trametinib, according to one embodiment.
  • Figure 5c is a bar graph showing the expression level of TRIM51 when several uveal melanoma cancer cell lines were treated with the MEK inhibitor selumetinib according to one embodiment.
  • 5D and 5E show before (pre-treatment) and after (post-treatment) treatment with a BRAF-MEK inhibitor in two independent melanoma biopsy samples (GSE65185 (FIG. 5D) and GSE99898 (FIG. 5E)), respectively, according to one embodiment.
  • ) is a paired plot showing the expression level of TRIM51.
  • 5F and 5G are paired plots showing the expression level of TRIM51 during initial (FIG. 5F) and progression (FIG. 5F) after treatment with a BRAF-MEK inhibitor, according to one embodiment.
  • Figure 6a is a graph showing the expression level of TRIM51 according to the disease progression and status of melanoma examined using four independent gene expression omnibus (GEO) data sets according to one embodiment.
  • GEO gene expression omnibus
  • 6B is a Kaplan-Meier curve showing overall and progression-free survival of TRIM51-high (z-score ⁇ 2) and TRIM51-low (z-score ⁇ 2) patients in cutaneous cutaneous melanoma (SKCM) according to one embodiment. it's a graph
  • Figure 6c shows total and no TRIM51-high ( ⁇ median expression level) and TRIM51-low ( ⁇ median expression level) patients of uveal melanoma (UVM) using The Cancer Genome Atlas (TCGA) data according to one embodiment. It is a Kaplan-Meier curve graph showing progression survival rate.
  • FIG. 7A shows mRNA expression levels of genes encoding granzymes and Lck in TRIM51-high ( ⁇ moderate level) and TRIM51-low (z-score ⁇ moderate level) patients with skin cutaneous melanoma (SKCM) according to one embodiment. is a graph showing
  • Figure 7B shows a significant association between the expression level of TRIM51 and CD8 T cell signature and three immunotherapy-related transcriptional signatures including signature down (Nivo_resistant_melanoma_down) and up-regulation (Nivo_resistant_melanoma_up) in nivolumab-resistant melanoma, according to one embodiment. It is a gene set enrichment analysis (GSEA) plot showing.
  • GSEA gene set enrichment analysis
  • variable includes all values within the stated range inclusive of the stated endpoints of the range.
  • a range of "5 to 10" includes values of 5, 6, 7, 8, 9, and 10, as well as any subrange of 6 to 10, 7 to 10, 6 to 9, 7 to 9, and the like. inclusive, as well as any value between integers that fall within the scope of the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like.
  • the range of "10% to 30%” includes values such as 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, as well as values from 10% to 15%, 12% to 12%, etc. It will be understood to include any sub-range, such as 18%, 20% to 30%, and the like, as well as any value between reasonable integers within the scope of the stated range, such as 10.5%, 15.5%, 25.5%, and the like.
  • BRAF inhibitors or MEK inhibitors have been recommended for conventional melanoma treatment, but melanoma patients show resistance, and accordingly, in order to improve the treatment rate of treatment for advanced melanoma patients, analysis of the interaction of melanoma cells is needed.
  • the establishment of a strategy based on melanoma resistance and the discovery of treatment predictive response markers for melanoma drugs were urgently needed.
  • the present inventors verified the effectiveness as a candidate for overcoming melanoma drug resistance through the control of TRIM51 mRNA or protein, and confirmed that it showed clinical significance as a predictive marker for treatment response.
  • melanoma It was confirmed that the derived TRIM51 is highly likely to be usefully used as a biomarker for predicting melanoma treatment response, and this was completed.
  • the biomarker for predicting melanoma drug resistance includes TRIM51 (Tripartite Motif-Containing 51) mRNA or protein.
  • the melanoma treatment includes one or more inhibitors selected from the group consisting of BRAF inhibitors and MEK inhibitors.
  • melanoma refers to a cutaneous malignant neoplasm (cancer) composed of melanocytes, and in one embodiment, the melanoma may be a BRAF mutant melanoma, the melanoma comprising cutaneous melanoma, and uveal It may be one or more melanomas selected from the group consisting of melanomas.
  • TriM51 used is an abbreviation of Tripartite Motif-Containing 51, and information on the gene and protein of TRIM51 can be easily identified through a known database such as NCBI gene bank, and the TRIM51 gene is, for example, SEQ ID NO: 1 It may be a polynucleotide consisting of the nucleotide sequence of or a polynucleotide consisting of the nucleotide sequence described in Gene ID: 84767 of the NCBI Genbank, and the TRIM51 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
  • the term "prediction" is used to refer to the likelihood that a subject patient will respond favorably or unfavorably to a drug or set of drugs.
  • the prediction relates to the extent of such response. For example, a prediction relates to whether and/or the probability that a patient will survive without melanoma recurrence after treatment, e.g., treatment with a particular therapeutic agent and/or surgical removal of a primary tumor and/or chemotherapy for a specified period of time. will be.
  • These predictions can be used clinically to make treatment decisions by selecting the most appropriate treatment modalities for melanoma patients.
  • the prediction is whether the patient will respond favorably to a therapeutic treatment, such as a given therapeutic treatment, e.g., administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc., or whether long-term survival of the patient is possible after the therapeutic treatment. It is a useful tool for forecasting.
  • a therapeutic treatment such as a given therapeutic treatment, e.g., administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc., or whether long-term survival of the patient is possible after the therapeutic treatment. It is a useful tool for forecasting.
  • predicting melanoma drug resistance means predicting whether a patient will respond preferentially or unfavorably to melanoma treatment, or predicting the risk of melanoma drug resistance. Therefore, if it is possible to predict patients for whom an effect can be expected (responders) and patients for whom no effect can be expected (non-responders) before the start of treatment, chemotherapy with high efficacy and safety can be realized.
  • the prediction method can be used clinically to make treatment decisions by selecting the most appropriate treatment modalities for patients with melanoma.
  • the composition for predicting melanoma drug resistance includes an agent for detecting TRIM51 (Tripartite Motif-Containing 51) mRNA or protein expression level as an active ingredient. At this time, if there is overlapping content with the biomarker for melanoma drug resistance prediction among the contents related to the composition for melanoma drug resistance prediction, it can be omitted.
  • TRIM51 Tripartite Motif-Containing 51
  • the expression level of TRIM51 can be measured to predict and diagnose melanoma treatment resistance.
  • the sequence can be modified to a certain extent in diagnosing the progression or onset of cancer.
  • a sequence in which 80% or more, specifically 90% or more, more specifically 95% or more, and even more specifically 98% homology is maintained by such artificial modification is the target in the present invention. It will be readily understood that it is equivalent to the above sequence of the present invention, as long as it can be used as a cancer diagnostic marker and allows a significant comparison of the expression level difference between a normal subject and a subject suspected of having cancer.
  • the agent for measuring the mRNA expression level is a primer or probe that specifically binds to TRIM51 mRNA
  • the agent for measuring the expression level of the protein is an antibody that specifically binds to the TRIM51 protein, a fragment thereof, or an aptamer. it can be
  • agent for measuring the expression level of TRIM51 mRNA refers to an agent used in a method for determining the expression of mRNA contained in a sample, preferably RT-PCR or competitive RT-PCR (Competitive RT -PCR), Real-time RT-PCR, RNase protection assay (RPA; RNase protection assay), Northern blotting, gene chip analysis, etc.
  • RT-PCR or competitive RT-PCR Competitive RT-PCR
  • RNase protection assay RNase protection assay
  • Northern blotting gene chip analysis, etc.
  • target genes used in methods It may be a primer or probe capable of binding to, but is not particularly limited thereto.
  • primer refers to a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and serving as a starting point for template strand copying. It refers to a short nucleic acid sequence that functions. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.
  • reagents for polymerization i.e., DNA polymerase or reverse transcriptase
  • probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundreds of bases in length that can form a specific binding with a gene or mRNA. It may be manufactured in the form of a single stranded DNA (DNA) probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection, but is not limited thereto.
  • DNA single stranded DNA
  • RNA probe or the like
  • the primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods.
  • Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologues of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoramidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.)
  • a nucleic acid may contain one or more additional covalently linked moieties, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalants (eg, acridine, psoralen, etc.). ), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.
  • Nucleic acid sequences of the present invention can also be modified with labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, and biotin.
  • agent for detecting the TRIM51 protein expression level is a process for confirming the presence and expression level of a protein expressed from a cancer marker gene in a biological sample in order to diagnose cancer, specifically, the protein of the gene
  • the amount of the protein can be confirmed using an antibody, a fragment thereof, or an aptamer that specifically binds to.
  • antibody refers to a specific protein molecule directed against an antigenic site.
  • antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal, monoclonal and recombinant antibodies. Since cancer marker proteins have been identified as described above, antibodies can be easily prepared using techniques well known in the art.
  • Polyclonal antibodies can be produced by a method well known in the art, in which the above-described colorectal cancer or prostate cancer marker protein antigen is injected into an animal and blood is collected from the animal to obtain serum containing the antibody.
  • Such polyclonal antibodies can be prepared from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, and cow.
  • Monoclonal antibodies can be prepared using a hybridoma method or phage antibody library technology well known in the art.
  • the antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
  • the antibodies include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains.
  • a functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.
  • aptamer refers to a polynucleotide composed of a special kind of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity.
  • DNA DNA, RNA, or modified nucleic acid
  • aptamers can specifically bind to antigenic substances in the same way as antibodies, but are more stable than proteins, have a simple structure, and are composed of polynucleotides that are easy to synthesize, so they can be used instead of antibodies.
  • a kit for diagnosing melanoma includes the composition for diagnosing melanoma.
  • the kit can be used to predict melanoma drug resistance by measuring the expression level of TRIM51 in a sample isolated from a melanoma-affected individual, but is not particularly limited thereto, Primers or probes for measuring the expression level of TRIM51 In addition, one or more other component compositions, solutions or devices suitable for the analytical method may be included.
  • the diagnostic kit for measuring the expression level of TRIM51 of the present invention may be a kit containing essential elements required to perform RT-PCR.
  • the RT-PCR kit contains, in addition to each primer pair specific for the gene, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase enzymes such as, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like.
  • dNTPs deoxynucleotides
  • Taq-polymerase reverse transcriptase enzymes
  • a primer pair specific to a gene used as a quantitative control may be included.
  • kits of the present invention may include essential elements required to perform gene chip analysis.
  • a gene chip analysis kit may include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, reagents, enzymes, and the like for preparing a fluorescently labeled probe.
  • the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.
  • the kit may include essential elements required to perform ELISA.
  • ELISA kits contain antibodies specific for marker proteins.
  • An antibody is an antibody that has high specificity and affinity for each marker protein and little cross-reactivity to other proteins, and is a monoclonal antibody, polyclonal antibody, or recombinant antibody.
  • ELISA kits may also include antibodies specific for a control protein.
  • Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg, conjugated with antibodies) and substrates thereof or those capable of binding the antibody. may contain other substances and the like.
  • a method for providing information necessary for predicting resistance to a melanoma therapeutic agent includes measuring the mRNA or protein level of TRIM51 in a sample isolated from a melanoma patient; Comparing the measured mRNA or protein level of TRIM51 with the level of TRIM51 mRNA or protein in a normal control sample; and determining that resistance to a melanoma treatment is low when the measured mRNA or protein level of TRIM51 is higher than that of the normal control sample.
  • sample isolated from a melanoma patient refers to the mRNA of the TRIM51 gene, which is a melanoma marker, or Samples such as tissues, cells, blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine having different protein levels thereof are included, but are not limited thereto.
  • the mRNA level can be measured in various ways, and specifically, primers or probes that specifically bind to the TRIM51 gene can be used.
  • the mRNA level is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time reverse transcriptase polymerase reaction (real time quantitative RT-PCR), RNase protection assay (RNase protection method), it may be measured by Northern blotting or gene chip, but is not limited thereto.
  • RT-PCR reverse transcriptase polymerase reaction
  • competitive RT-PCR competitive reverse transcriptase polymerase reaction
  • real time reverse transcriptase polymerase reaction real time quantitative RT-PCR
  • RNase protection method RNase protection method
  • the TRIM51 protein level measurement method includes Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc. There is, but is not limited to this.
  • antigen-antibody complex refers to a combination of a melanoma marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be quantitatively measured through the size of the signal of a detection label do.
  • detection labels may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but are not necessarily limited thereto.
  • enzymes include ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Theranase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphophenolpyruvate deca voxylase, ⁇ -latamase, and the like, but are not limited thereto.
  • Fluorescent substances include fluorescein, isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanine, o-phthalaldehyde, fluorescamine, and the like, but are not limited thereto.
  • Ligands include biotin derivatives and the like, but are not limited thereto.
  • Luminescent substances include acridinium ester, luciferin, luciferase, and the like, but are not limited thereto.
  • Microparticles include, but are not limited to, colloidal gold, colored latex, and the like.
  • Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K 4 W(CN) 8 ,[Os(bpy) 3 ] 2+ , [RU(bpy) 3 ] 2+ , [MO(CN) 8 ] 4- , and the like, but are not limited thereto.
  • Radioisotopes include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co , 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re. .
  • ELISA includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support, followed by labeled antibody that recognizes this antibody It includes various ELISA methods such as indirect sandwich ELISA using secondary antibodies.
  • a labeled antibody recognizing the antigen of the antigen-antibody complex is attached to enzymatically develop color, or for the antibody recognizing the antigen of the antigen-antibody complex It is detected by a sandwich ELISA method in which a labeled secondary antibody is attached to enzymatically develop color. Resistance to the melanoma treatment can be confirmed by checking the degree of complex formation between the cancer marker protein and the antibody.
  • Western blotting using one or more antibodies against the melanoma marker is used.
  • Total proteins are separated from the sample, subjected to electrophoresis to separate the proteins according to size, and transferred to a nitrocellulose membrane to react with antibodies.
  • Cancer occurrence can be confirmed by checking the amount of protein produced by gene expression by a method of checking the amount of the produced antigen-antibody complex using a labeled antibody.
  • the detection method consists of a method of examining the expression level of a marker gene in a control group and the expression level of a marker gene in cancerous cells.
  • mRNA or protein levels can be expressed as absolute (eg, ⁇ g/ml) or relative (eg, relative intensity of signal) differences in the marker proteins described above.
  • immunohistochemical staining is performed using at least one antibody against the melanoma marker.
  • a paraffin-embedded block is prepared by a method well known in the art. These are made into slices with a thickness of several ⁇ m and attached to a glass slide, and then reacted with one selected from the above antibodies by a known method. Thereafter, the unreacted antibody is washed, labeled with one of the above-mentioned detection labels, and whether the antibody is labeled is read under a microscope.
  • a protein chip in which one or more antibodies against the melanoma marker are arranged at a predetermined location on a substrate and immobilized at a high density is used.
  • a method of analyzing a sample using a protein chip isolates a protein from the sample, hybridizes the isolated protein with the protein chip to form an antigen-antibody complex, reads it, and confirms the presence or expression level of the protein, Resistance to melanoma treatment can be confirmed.
  • a method for screening a melanoma therapeutic agent responsiveness enhancer includes contacting a test substance with a sample separated from a melanoma patient; Measuring the mRNA or protein level of TRIM51 in a sample contacted with the test material; and selecting a test substance having a reduced mRNA or protein level of TRIM51 compared to a control sample.
  • the candidate substance A substance that reduces the expression level of the TRIM51 of the present invention when present than the level in the absence of the candidate substance can be predicted as a melanoma therapeutic agent responsiveness enhancer.
  • 'candidate' refers to an unknown candidate material used in screening to examine whether or not it affects the expression level of a gene or affects the expression or activity of a protein.
  • the samples include, but are not limited to, chemical substances, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.
  • CCL cancer cell line
  • CTRP Cancer Therapeutics Response Portal
  • PRISM Profile Relative Inhibition Simultaneously in Mixtures
  • TCGA Cancer Genome Atlas
  • cBioPortal for clinical data of melanoma patients.
  • CTRP data was used to quantify the effect of each of the 481 compounds by calculating the area under a 16-point concentration-response curve (AUC) across 827 CCLs.
  • AUC concentration-response curve
  • PRISM data compared to the negative control at a single concentration
  • the fold change (log 2 ) of chemical perturbation viability of the pooled 578 CCLs was estimated.
  • GEO Gene Expression Omnibus
  • GSE127988 (13) Transcripts of A375 melanoma cells treated with 7 doses of vemurafenib (0.001-10 uM) for 24 hours.
  • GSE127948 (14): transcript of 92.1 uveal melanoma cells treated with trametinib (10 nM) for 24 hours.
  • GSE33655 (15): Transcriptome of uveal melanoma cells treated with selumetinib for 8 hours.
  • N T Total number of drugs tested (481 drugs from CTRP data).
  • N R The number of drugs with a positive correlation greater than X between the expression level of "Gene A” and drug sensitivity.
  • NS The number of drugs with a negative correlation lower than -X between the expression level of "Gene A” and drug sensitivity.
  • DRS(0.3)_gene A is ⁇ -0.3 between the expression level of "gene A” and drug sensitivity in the number of drugs with a correlation >0.3 between the expression level of "gene A” and drug sensitivity. It is the number minus the number of drugs with a correlation, divided by 481 drugs.
  • a given gene with a high DRS can be classified as a predominantly resistance-related gene for 481 small molecules, whereas a gene with a low DRS can be classified as a predominantly susceptibility-related gene.
  • genes with relatively high DRS are likely to be resistant to most small molecules tested, regardless of development status (e.g., FDA-approved, clinical investigations, and molecular probes) and target class (e.g., kinases). .
  • Figure 1a shows the genes aligned with the drug response score (DRS) converted to DRS (0.3) by calculating the pharmacologic correlation of each gene for 481 drugs of Cancer Therapeutics Response Portal (CTRP) data according to one embodiment. is the graph shown.
  • DRS drug response score
  • CRP Cancer Therapeutics Response Portal
  • Figure 1b is a graph showing the relative display of DRS for all drugs, kinase inhibitors and BRAF inhibitors according to one embodiment.
  • the DRS to BRAF inhibitors showed different behavior. That is, it was confirmed that genes with high DRS for all drugs or BRAF inhibitors other than kinase inhibitors were not biased toward resistance or sensitivity to most drugs.
  • BRAF inhibitors ie, BRAF gene or protein inhibitors, are highly likely to identify specific sensitivity-related genes.
  • Example 2 Analysis of TRIM51 gene expression level and susceptibility to BRAF-MEK inhibitors
  • Figure 2 is a graph showing the TRIM51 mRNA level test using cBioPortal in various cancer cell lines (CCL).
  • FIG. 3A is a heat map of drug sensitivity profiles for several BRAF-MEK inhibitors as measured by area under the drug response curve (AUC) according to one embodiment
  • FIG. Figure 3c is a diagram showing the expression level of BRAF-MEK pathway-related proteins measured by reverse-phase protein array (RPPA) displayed in 201 cancer cell lines (CCL) sorted by TRIM51 mRNA level according to one embodiment. is the diagram shown. (At this time, parentheses indicate TRIM51 mRNA level and Pearson's correlation coefficient)
  • RPPA reverse-phase protein array
  • the BRAF gene or protein inhibitor may include one or more selected from the group consisting of vemurafenib and dabrafenib, and the MEK gene or protein inhibitor may include trametinib can include
  • TRIM51-high CCL in which the BRAF gene or protein inhibitor-MEK gene or protein inhibitor is very effective, has a relatively high concentration of BRAF mutations (FIG. 3b), and BRAF, MEK and ERK1/ It was confirmed that it occurred together with an increased phosphorylation level of 2 (FIG. 3c).
  • FIGS. 4A and 4B show in a BRAF mutant melanoma cancer cell line (CCL) using Cancer Therapeutics Response Portal (CTRP) data (FIG. 4A) and simultaneous relative suppression profiling (PRISM) data in the mixture (FIG. 4B).
  • CTL Cancer Therapeutics Response Portal
  • PRISM simultaneous relative suppression profiling
  • Results were then validated using an independent data set from PRISM examining the sensitivity profiles of 4,518 drugs in 578 CCLs according to FIG. 4B. As a result, it was also confirmed that there was a significant correlation between the expression level of TRIM51 in BRAF-mutant melanoma and the sensitivity to BRAF MEK gene or protein inhibitors.
  • TRIM51 can be included in the BRAF-MEK signaling pathway, but also the expression level of TRIM51 can be expressed in response to BRAF-MEK gene or protein inhibitors even in BRAF mutant melanoma patients. It was confirmed that it can be used for detailed classification of characters.
  • Example 2 Analysis of pharmacological activity related to TRIM51 gene expression level and BRAF-MEK inhibitor
  • Example 1 the fact that the basal expression level of TRIM51 was associated with sensitivity to multiple inhibitors of both BRAF and MEK confirmed that the expression level of TRIM51 could be involved in the BRAF-MEK signaling pathway.
  • 5a to 5g are graphs showing the expression level of TRIM51, a surrogate marker for the pharmacological activity of a BRAF-MEK inhibitor, according to one embodiment.
  • FIG. 5a is a bar graph showing the expression level of TRIM51 when A375 melanoma cells were treated with the BRAF inhibitor vemurafenib according to one embodiment.
  • Figure 5b is a bar graph showing the expression level of TRIM51 when 92.1 uveal melanoma cells were treated with the MEK inhibitor, trametinib, according to one embodiment.
  • Figure 5c is a bar graph showing the expression level of TRIM51 when several uveal melanoma cancer cell lines were treated with the MEK inhibitor selumetinib according to one embodiment.
  • 5D and 5E show before (pre-treatment) and after (post-treatment) treatment with a BRAF-MEK inhibitor in two independent melanoma biopsy samples (GSE65185 (FIG. 5D) and GSE99898 (FIG. 5E)), respectively, according to one embodiment.
  • ) is a paired plot showing the expression level of TRIM51.
  • 5F and 5G are paired plots showing the expression level of TRIM51 during initial (FIG. 5F) and progression (FIG. 5F) after treatment with a BRAF-MEK inhibitor, according to one embodiment.
  • Example 3 TRIM51 gene expression level and survival and immune tolerance analysis of melanoma patients
  • FIG. 6A is a graph showing the expression level of TRIM51 according to the disease progression and status of melanoma examined using four independent gene expression omnibus (GEO) data sets according to one embodiment.
  • GEO gene expression omnibus
  • Figure 6b is a Kaplan-Meier curve showing overall and progression-free survival of TRIM51-high (z-score ⁇ 2) and TRIM51-low (z-score ⁇ 2) patients in cutaneous cutaneous melanoma (SKCM) according to one embodiment. it's a graph
  • Figure 6c shows total and no TRIM51-high ( ⁇ median expression level) and TRIM51-low ( ⁇ median expression level) patients of uveal melanoma (UVM) using The Cancer Genome Atlas (TCGA) data according to one embodiment. It is a Kaplan-Meier curve graph showing progression survival rate.
  • a data set from an independent study showed that the expression level of TRIM51 was generally upregulated during disease progression, and the mRNA level of TRIM51 was significantly increased in both primary and metastatic melanoma compared to normal nevi. could confirm that
  • GSEA gene set enrichment analysis

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Abstract

La présente invention concerne un biomarqueur TRIM51 permettant de prédire la résistance au traitement contre le mélanome et son utilisation. Après avoir vérifié son efficacité en tant que candidat pour surmonter la résistance aux médicaments du mélanome par la régulation de l'ARNm ou de la protéine TRIM51, et confirmé sa signification clinique en tant que marqueur de prédiction de la réponse au traitement, TRIM51 dérivé du mélanome est très susceptible de trouver des applications avantageuses en tant que biomarqueur de prédiction de la réponse au traitement contre le mélanome.
PCT/KR2022/011017 2022-02-07 2022-07-27 Biomarqueur trim51 pour prédire la résistance au traitement contre le mélanome et son utilisation WO2023149608A1 (fr)

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KR20180099123A (ko) * 2017-02-28 2018-09-05 부산대학교 산학협력단 항암제에 대한 저항성 예측용 바이오마커
KR20210114219A (ko) * 2020-03-10 2021-09-23 연세대학교 산학협력단 약제 내성 진단용 바이오 마커 조성물 및 이를 이용한 진단 키트
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