WO2023149608A1 - Trim51 biomarker for predicting melanoma treatment resistance and use thereof - Google Patents

Abstract

The present invention relates to a TRIM51 biomarker for predicting melanoma treatment resistance and a use thereof. As a result of verifying the effectiveness as a candidate for overcoming melanoma drug resistance through the regulation of TRIM51 mRNA or protein, and confirming whether it shows clinical significance as a treatment response prediction marker, melanoma-derived TRIM51 is very likely to find advantageous applications as a biomarker for predicting melanoma treatment response.

Description

TRIM51 biomarker for predicting melanoma treatment resistance and its uses

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.

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.

Accordingly, 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.

The purpose of solving the above problem is as follows.

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) For the purpose of providing 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.

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 the 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; aims to do

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 material; And compared to a control sample, 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.

In this case, 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.

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 according to another aspect 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 according to another aspect 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; As a method for providing information necessary for predicting resistance to a melanoma treatment, 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 according to another aspect 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.

That is, as a result of verifying the effectiveness as a melanoma drug resistance overcoming candidate through the control of the TRIM51 mRNA or protein, and confirming whether it shows clinical significance as a treatment response prediction marker, 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.

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).

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, and 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)

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). A graph showing the correlation between expression levels and susceptibility to BRAF (dabrafenib and GDC-0879) and MEK (PD318088 and trametinib) gene or protein inhibitors.

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.

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.

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.

The above objects, other objects, features and advantages will be easily understood through the following preferred embodiments in conjunction with the accompanying drawings. However, it is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed content will be thorough and complete and the technical idea will be sufficiently conveyed to those skilled in the art.

Like reference numerals have been used for like elements throughout the description of each figure. In the accompanying drawings, the dimensions of the structures are shown enlarged than actual for clarity of the present invention. Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise.

In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that it does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof. In addition, when a part such as a layer, film, region, plate, etc. is said to be "on" another part, this includes not only the case where it is "directly on" the other part, but also the case where another part is present in the middle. Conversely, when a part such as a layer, film, region, plate, etc. is said to be "under" another part, this includes not only the case where it is "directly below" the other part, but also the case where another part is in the middle.

Unless otherwise specified, all numbers, values and/or expressions expressing quantities of components, reaction conditions, polymer compositions and formulations used herein refer to the number of factors that such numbers arise, among other things, to obtain such values. Since these are approximations that reflect the various uncertainties of the measurement, they should be understood to be qualified by the term "about" in all cases. Also, when numerical ranges are disclosed herein, such ranges are contiguous and include all values from the minimum value of such range to the maximum value inclusive, unless otherwise indicated. Furthermore, where such ranges refer to integers, all integers from the minimum value to the maximum value inclusive are included unless otherwise indicated.

In this specification, where ranges are stated for a variable, it will be understood that the variable includes all values within the stated range inclusive of the stated endpoints of the range. For example, 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. Also, for example, 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.

Accordingly, as a result of intensive research to solve the above problems, 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. As a result, 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 according to one embodiment includes TRIM51 (Tripartite Motif-Containing 51) mRNA or protein.

Preferably, the melanoma treatment includes one or more inhibitors selected from the group consisting of BRAF inhibitors and MEK inhibitors.

The term "melanoma", as used, 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.

The term "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.

As used herein, 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. In one embodiment, 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.

As used herein, the term "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. In addition, the prediction method can be used clinically to make treatment decisions by selecting the most appropriate treatment modalities for patients with melanoma.

In addition, the composition for predicting melanoma drug resistance according to another embodiment 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.

Based on the sequence of TRIM51, 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. For those skilled in the art, 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.

Specifically, 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 is an antibody that specifically binds to the TRIM51 protein, a fragment thereof, or an aptamer. it can be

The term "agent for measuring the expression level of TRIM51 mRNA" as used herein 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. Specific to target genes used in methods It may be a primer or probe capable of binding to, but is not particularly limited thereto.

The term "primer" as used herein 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.

As used herein, the term "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.

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.)

There is a variation of 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.

The term "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.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For purposes of this purpose, 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.

In addition, 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.

As an analysis method for confirming the amount of the target protein bound to the antibody using the antibody, Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and Ou Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein Chips (protein chips) and the like, but are not limited thereto.

The term "aptamer" as used herein 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. means a kind of As described above, 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. can

A kit for diagnosing melanoma according to another embodiment 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.

Specifically, 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. In addition, a primer pair specific to a gene used as a quantitative control may be included.

In addition, specifically, the kit 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. In addition, the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

In addition, specifically, 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.

In addition, a method for providing information necessary for predicting resistance to a melanoma therapeutic agent according to another embodiment 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.

The process of isolating mRNA from a sample isolated from a melanoma patient can be performed using a known process, and the term "sample isolated from a melanoma patient" as used in the present invention 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.

In addition, specifically, 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.

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.

Through the above analysis methods, it is possible to compare the amount of antigen-antibody complex formation in normal controls and the amount of antigen-antibody complex formation in melanoma suspected patients, and a significant increase in the expression level of melanoma marker genes to proteins It is possible to diagnose whether a patient with melanoma is resistant to melanoma treatment by determining whether or not the patient has melanoma.

The term "antigen-antibody complex" as used herein 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.

These 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. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-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, β-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. .

The protein expression level measurement is specifically using the ELISA method. 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. More preferably, after attaching the antibody to the solid support and reacting the sample, 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.

Also, specifically, 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, μg/ml) or relative (eg, relative intensity of signal) differences in the marker proteins described above.

Also, specifically, immunohistochemical staining is performed using at least one antibody against the melanoma marker. After collecting and fixing normal colonic epithelial tissue and tissue suspected of cancer, a paraffin-embedded block is prepared by a method well known in the art. These are made into slices with a thickness of several μm 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.

In addition, more specifically, 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 according to another embodiment 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.

After measuring the level of TRIM51 of the present invention in cells expressing the TRIM51 gene in the absence of the candidate substance and also measuring the level of the TRIM51 of the present invention in the presence of the candidate substance and comparing the two, 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.

The term 'candidate' as used herein 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.

The present invention will be described in more detail through the following examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Experimental Example: Data Analysis and Statistical Analysis

1. Data analysis

For the analysis of the following examples, the following database was utilized.

- Cancer Cell Line Encyclopedia (CCLE) for multiomics data analysis of cancer cell line (CCL)

- CTRP (Cancer Therapeutics Response Portal) PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) profiling for CCL's drug sensitivity profile data

- Cancer Genome Atlas (TCGA) and cBioPortal for clinical data of melanoma patients.

Specifically, the 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. In addition, the 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.

Additionally, to identify genes associated with drug sensitivity, we investigated the Pearson correlation coefficient between log ₂ -transformed gene expression levels (from CCLE data) and AUC (from CTRP data) or sensitivity fold change (from PRISM data). .

In addition, we used the following Gene Expression Omnibus (GEO) data set to investigate the mRNA level of TRIM51 according to melanoma disease status upon treatment with BRAF gene or protein inhibitor-MEK gene or protein inhibitor.

(I) GSE127988 (13): Transcripts of A375 melanoma cells treated with 7 doses of vemurafenib (0.001-10 uM) for 24 hours.

(II) GSE127948 (14): transcript of 92.1 uveal melanoma cells treated with trametinib (10 nM) for 24 hours.

(III) GSE33655 (15): Transcriptome of uveal melanoma cells treated with selumetinib for 8 hours.

(IV) GSE65185 (16): Transcripts of melanoma tumors (n=19) biopsied before treatment (vemurafenib or dabrafenib) and during disease progression.

(V) GSE99898 (17): biopsy before (n=17) and after (n=8) treatment (vemurafenib, dabrafenib or dabrafenib plus trametinib) and during disease progression (n=13) transcriptome of melanoma tumors.

(VI) GSE15605 (18): transcripts of normal skin (n=16), primary (n=46) and metastatic melanoma (n=12).

(VII) GSE112509 (19): Transcripts of microdissected melanocytic nevus (n=23) and primary melanoma (n=57).

(VIII) GSE83583 (20): Transcripts of melanocytes (n=3) and primary melanoma (n=32).

(IX) GSE12391 (21): Common melanocytic nevus (n=18), primary radial anagen melanoma (n=8), primary vertical anagen melanoma (n=15) and metastasis of melanoma (n=5). corpse.

The following examples were carried out in accordance with the Declaration of Helsinki (revised in 2013).

2. Statistical analysis

Figures were generated and statistical analysis was performed using GraphPad Prism (version 9.0; GraphPad Software, San Diego, CA, USA).

A t-test was performed to compare the means between the two groups, and Pearson's correlation coefficient was calculated to evaluate the correlation.

For Kaplan-Meier survival analysis, patients were classified into TRIM51-high and TRIM51-low according to z-score (≥2) or median expression level, and statistical significance between the two groups was investigated by log-rank test. At this time, statistical significance was set at P<0.05.

Example 1: Analysis of Melanoma Expression Genes by Drug Response Score (DRS)

Well-defined CTRP data including sensitivity profiles of 860 CCLs to 481 small molecules were analyzed to evaluate the pharmacotransfer properties of the gene.

In order to quantitatively measure the relative transcriptional propensity of each gene for resistance or susceptibility to a total of 481 small molecules investigated, a scoring metric that evaluates each gene based on the correlation between gene expression and drug sensitivity, or DRS, was adopted. .

[Equation 1]

X: The absolute value of the correlation coefficient X.

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.

For example, "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.

Thus, 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.

According to Ordered DRS, 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). .

In addition to the broad predisposition of genes for susceptibility or resistance, we focused on DRS for selective inhibitors targeting specific proteins.

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.

Referring to Figure 1a, it was confirmed that genes having high DRS for most small molecules showed high DRS for selective inhibitors of PI3Kβ, ALK, or MET.

Figure 1b is a graph showing the relative display of DRS for all drugs, kinase inhibitors and BRAF inhibitors according to one embodiment.

Referring to FIG. 1B, 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.

Through this, it was confirmed that 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).

Referring to Figure 2 above, since high TRIM51 mRNA levels with a z-score of 2≥ were found only in melanoma CCL (top), exclusively high mRNA expression of TRIM51 was also found in cutaneous melanoma CCL by box plot (bottom). was able to confirm what was displayed. That is, we focused on TRIM51, a gene that is exclusively highly expressed in melanoma-derived CCL, where BRAF inhibitors are primarily utilized as therapeutics.

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, and 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)

Referring to Figure 3a, it was confirmed that increased TRIM51 levels in 201 CCLs with TRIM51 transcript detection were clearly correlated with improved efficacy of multiple BRAF and MEK inhibitors, as indicated by reduced AUC.

In addition, 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

At this time, referring to the above figure, TRIM51-high CCL (FIG. 3a), 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).

Therefore, to rule out the possibility that the high efficacy is due to the dependence of BRAF mutation, we investigated the association of TRIM51 expression levels with sensitivity to the BRAF gene or protein inhibitor-MEK gene or protein inhibitor using CCL restricted to BRAF mutant melanoma. The results thereof are shown in FIGS. 4a and 4b.

Specifically, 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). , A graph showing the correlation between expression levels of TRIM51 and susceptibility to BRAF (dabrafenib and GDC-0879) and MEK (PD318088 and trametinib) genes or protein inhibitors.

Referring to Figure 4a, there is a strong correlation between the expression level of TRIM51 and sensitivity to multiple BRAFs (eg dabrafenib and GDC-0879) and MEK gene or protein inhibitors (eg PD318088 and trametinib). You can check.

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.

That is, according to the above results, not only can it be confirmed that the expression level of 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

According to 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.

Specifically, 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.

Referring to Figure 5a, when treated with the BRAF gene or protein inhibitor vemurafenib, it was confirmed that the expression level of TRIM51 was dramatically reduced in a dose-dependent manner in melanoma CCL, A375 even at a concentration of less than 100 nM.

Referring to Figures 5b and 5c, when treated with the MEK gene or protein inhibitor trametinib or selumetinib, it was confirmed that the expression level of TRIM51 was significantly reduced in various uveal melanoma CCL.

Through this, it was confirmed that the BRAF-MEK pathway can positively regulate the mRNA level of TRIM51 in melanoma.

In addition, to demonstrate the clinical relevance of BRAF-MEK pathway-dependent transcriptional regulation of TRIM51, according to Figures 5D to 5F, we measured the mRNA level of TRIM51 between paired melanoma biopsies before and after treatment with a BRAF inhibitor. compared.

Specifically, referring to Figure 5d, it was confirmed that the expression level of TRIM51 was consistently reduced in 11 out of 12 patients investigated.

In addition, referring to FIG. 5E, early treatment with a BRAF-MEK gene or protein inhibitor (vemurafenib, dabrafenib or dabrafenib and trametinib combination therapy) was also independently tested in 8 pairs of melanoma biopsy samples. It was confirmed that the expression level of TRIM51 was stably reduced in 6 of them.

In addition, referring to Figure 5f, it can be confirmed that the mRNA level of TRIM51 recovered or even increased in 3 out of 4 pairs of melanoma biopsies after disease progression to the inhibitor, and referring to Figure 5g, the mRNA level of TRIM51 after progression It was confirmed that out of 13 pairs of samples, 3 patients increased, 4 patients did not change, and 6 patients decreased.

Taken together, we demonstrate the positive regulation of TRIM51 mRNA levels by the BRAF-MEK pathway and show that the expression levels of TRIM51 can faithfully represent the degree of activity and resistance to BRAF-MEK inhibitors after treatment in both in vitro and clinical situations. I was able to confirm.

Example 3: TRIM51 gene expression level and survival and immune tolerance analysis of melanoma patients

Whether the expression level of TRIM51 is related to melanoma disease progression and prognosis was evaluated, and the results are shown in FIGS. 6a to 6c.

Specifically, 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.

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.

Referring to FIG. 6A , 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

In addition, referring to Figures 6b and 6c, it was confirmed that the upregulation of TRIM51 mRNA level was significantly associated with low survival rates in both cutaneous and uveal melanomas.

In addition, the importance of immunotherapy for melanoma was investigated to determine whether the poor survival of patients with high TRIM51 was associated with a poorer tumor microenvironment, especially immune resistance, by examining the association between the expression level of TRIM51 and immune resistance-related features. is shown in Figures 7a and 7b.

Referring to FIG. 7a , it was confirmed that the expression level of the gene encoding granzyme, a T cell effector molecule, was reduced in patients with high levels of TRIM51. That is, it was confirmed that patients with high TRIM51 showed low mRNA levels of LCK, which is a T cell dominant marker, indicating low T cell infiltration.

In addition, referring to FIG. 7B , gene set enrichment analysis (GSEA) confirmed that patients with a high level of TRIM51 tended to have a low level of CD8 T cell activity. In addition, it was confirmed that the expression pattern of patients with high TRIM51 showed a transcriptional bias toward resistance to nivolumab.

Taken together, we could confirm that patients with high TRIM51 may be more likely to be resistant to immunotherapy and their poor survival is partly due to immune tolerance features.

Claims (12)

1. TRIM51 (Tripartite Motif-Containing 51) As a biomarker for predicting melanoma drug resistance including mRNA or protein,

   The melanoma therapeutic agent is a biomarker, characterized in that it comprises one or more inhibitors selected from the group consisting of BRAF inhibitors and MEK inhibitors.
2. According to claim 1,

   The melanoma is a BRAF mutant melanoma, a biomarker for predicting melanoma drug resistance.
3. According to claim 1,

   The melanoma is one or more melanomas selected from the group consisting of cutaneous melanoma and uveal melanoma, a biomarker for predicting melanoma drug resistance.
4. According to claim 1,

   The resistance is intrinsic resistance (Intrinsic resistance), a biomarker for predicting melanoma therapeutic resistance.
5. TRIM51 (Tripartite Motif-Containing 51) A composition for predicting resistance to a melanoma treatment comprising an agent measuring the mRNA or protein expression level as an active ingredient,

   The melanoma treatment agent is characterized in that it comprises one or more inhibitors selected from the group consisting of BRAF inhibitors and MEK inhibitors, composition.
6. According to claim 5,

   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 composition for predicting melanoma treatment resistance.
7. A kit for predicting resistance to a melanoma therapeutic agent comprising the composition for predicting resistance to a melanoma therapeutic agent of claim 5.
8. According to claim 7,

   A kit for predicting melanoma drug resistance, which is an RT-PCR kit, a DNA chip kit, or a protein chip kit.
9. 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 the melanoma treatment is low when the measured mRNA or protein level of TRIM51 is higher than the level of the normal control sample; As a method of providing information necessary for predicting resistance to a melanoma treatment,

   The method for providing information, characterized in that the melanoma therapeutic agent comprises at least one inhibitor selected from the group consisting of a BRAF inhibitor and a MEK inhibitor.
10. According to claim 9,

    The step of measuring the mRNA level of TRIM51 is to use a primer or probe that specifically binds to the TRIM51 mRNA.
11. According to claim 9,

    The step of measuring the TRIM51 protein level is to use an antibody, a fragment thereof, or an aptamer that specifically binds to the protein.
12. 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 material; and

    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 compared to a control sample,

    The screening method, characterized in that the melanoma therapeutic agent comprises at least one inhibitor selected from the group consisting of a BRAF inhibitor and a MEK inhibitor. The melanoma therapeutic agent comprises at least one inhibitor selected from the group consisting of a BRAF inhibitor and a MEK inhibitor. Characterized in that it comprises, a screening method.

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