KR20150085459A - Novel NTRK1 fusion gene as colon cancer marker and the uses thereof - Google Patents

Abstract

The present invention relates to a fusion protein of lamin A (LMNA) or tropomyosin 3 (TPM3) and neurotrophic tyrosine kinase receptor type 1 (NTRK1), and a fusion gene polynucleotide encoding the same. The present invention also relates to a colon cancer diagnosis composition including the fusion protein, the fusion gene, or a formulation for measuring a transcription product of the fusion gene, to a kit for diagnosing colon cancer including the same, to a method for providing information needed for colon cancer diagnosis, which includes a step of measuring a level of the NTRK1 fusion gene, a transcription product thereof, or protein thereof, and to a method for providing information needed to make prognosis of colon cancer treatment. In addition, the present invention relates to a method for screening a colon cancer treating agent including a step of measuring an expression level of the NTRK1 fusion gene, and to a pharmaceutical composition including an NTRK1 fusion protein inhibiting agent as an active ingredient for preventing or treating colon cancer. According to the present invention, provided is a diagnosis marker specific to colon cancer, thus it is possible to accurately diagnose colon cancer, to predict of clinical prognosis by classifying colon cancer patients, to predict efficacy of colon cancer treatment, and especially to predict efficacy of colon cancer treatment by the NTRK1 protein inhibiting agent. Accordingly, administration of an agent to a colon cancer patient for whom administration of an agent is considered to be ineffective can be limited. Therefore, efficient and safe colon cancer treatment can be realized.

Description

A novel NTRK1 fusion gene as a colon cancer marker and its use {Novel NTRK1 fusion gene as colon cancer marker and the uses thereof}

A fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) with NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the same. Further, the present invention relates to a composition for diagnosing colorectal cancer comprising the fusion protein, the fusion gene, or an agent for measuring the transcription product of the fusion gene, and a kit for diagnosing colorectal cancer comprising the same. In addition, the present invention relates to a method for providing information necessary for diagnosis of colon cancer, including the step of measuring the level of the NTRK1 fusion gene, its transcription product or its protein, and a method for providing information necessary for predicting colon cancer treatment prognosis. Further, the present invention relates to a method for screening a therapeutic agent for colorectal cancer, which comprises the step of measuring the expression level of the NTRK1 fusion gene, and a pharmaceutical composition for preventing or treating colorectal cancer comprising the NTRK1 fusion protein inhibitor as an active ingredient.

Colon cancer is the second most common cancer in cancer deaths, accounting for about 13% of all cancers. In the United States, about 50,310 people die of colorectal cancer annually and 136,830 new colorectal cancer cases are reported to occur. In Korea, colon cancer accounts for the fourth place in lung cancer, liver cancer, and stomach cancer in terms of mortality. In 1980, 5.8% of total cancer cases showed a steadily increasing trend in 6.15% in 1985, 8.2% in 1995, 11.2% in 2002 and 12.9% in 2012. Compared with 1999 data In 2011, mortality from gastric cancer, liver cancer and cervical cancer decreased, while mortality from colon cancer increased by 5.6%.

The 5 - year survival rate of all colorectal cancer patients is reported to be about 64.7% due to early diagnosis, curative surgery and chemotherapy using colonoscopy. However, the 5-year survival rate of primary colorectal cancer patients was 89.8% and the 5-year survival rate of second-stage colorectal cancer patients was improved to 70.5%, while the 5-year survival rate of end stage colorectal cancer patients was 12.9% , And it is difficult to improve the survival rate through existing therapies. The duration of disease-free survival of end-stage colorectal cancer patients treated with targeted therapies such as Erbitux (Merck) and Avastin (Roche), which recently received insurance benefits, was 0.9 months (p = 0.048) and 1.4 months = 0.0023), indicating that clinical efficacy is inadequate. Compared with breast cancer and lung cancer, colon cancer has not been studied in genomic studies, and thus, there is not much stratification of the molecular level. Therefore, in order to increase the survival rate of patients with colorectal cancer, it is urgent to stratify the molecular level and to find a new target for colon cancer.

It is known that various genetic changes such as various kinds of cancer genes and tumor suppressor genes are involved in the development of colon cancer. Actually, colorectal cancer is one of the most common genetic changes in carcinogenesis. In the case of colorectal cancer, the change of one cancer gene or tumor suppressor gene can not induce cancer alone. In order for normal colon mucosal cells to progress to colorectal cancer through adenoma stage, Changes in related genes should be accumulated, which is called multistep change of genes in the development of colorectal cancer. What is important here is not the sequence of gene changes at each step but the sum of the changes of genes that ultimately accumulate. The genetic changes involved in colon cancer development involve the mutation of cancer gene and tumor suppressor gene, DNA methylation abnormality, and mutation of DNA repair gene.

Since the formation of cancer is caused by a complex combination of various genes and the interaction of these genes, a genetic approach to discover new cancer genes and therapeutic targets has been made by investigating and comparing the expression and mutation of whole genes . Genes that specifically increase or decrease expression in cancer cells are thought to be involved in the survival and proliferation of cancer cells such as cell division, cell signaling, cytoskeleton, cell movement, cell defense, gene and protein expression, and intracellular metabolism . The development of cancer genomics has contributed to the stratification of patients such as breast cancer and lung cancer, and to the development of targets for tailored treatment for individual patients. However, the absence of an assessment of the clinical efficacy of the identified target has been evaluated as a major cause of failure in clinical studies of customized chemotherapeutic drugs developed through cancer genomics.

The NTRK1 fusion gene has been reported across a variety of carcinomas. The fusion gene of TPM3-NTRK1 has been reported in colon cancer and papillary thyroid carcinoma (Martin-Zanca et al., Nature, 1986, 319, 743-748; Wilton et al., Cytogenetics and cell genetics, 124), and myosin phosphatase Rho interacting protein (MPRIP) or CD74-NTRK1 fusion gene has been reported in lung cancer (Vaishnave et al., Nature medicine, 2013, vol. 19, p. 1469-1472; Patent No. WO 2014071358 A2), TP53 or lamin A / C: LMNA) -NTRK1 fusion genes have been reported in Spitzoid melanomas (Wiesner et al., Nature communications, vol. 5, 3116, 2014; RAB GTPase activating protein 1-like (RABGAP1L) -TRK1 fusion gene has been reported in gallbladder carcinoma (Ross et al., The oncologist, 2014, vol. 19, 242 pages), and in multifocal subspecies neurofascin NFASC) or brevican (BCAN) -NTRK1 fusion genes have been reported (Kim et al., PloS one, 2014, vol. 9, e91940). However, the role of the NTRK1 fusion gene as a cancer gene has been elucidated in some carcinomas, while the role, frequency, and clinical significance of the NTRK1 fusion gene as a cancer gene in colorectal cancer has not been elucidated.

The present inventors have conducted intensive research for genome-wide study of colon cancer through stratification of colorectal cancer patients, prediction of prognosis, and discovery of genes that can be targeted for treatment. As a result, LMNA-NTRK1 and TPM3- The NTRK1 fusion gene and its expression are significantly increased in certain patient groups, and the NTRK1 fusion gene and the fusion transcription product and fusion protein encoded thereby are the factors that determine the clinical prognosis of colorectal cancer. , Thereby completing the present invention.

One object of the present invention is to provide a fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) with NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the fusion protein.

Another object of the present invention is to provide a composition for diagnosing colorectal cancer comprising the fusion protein, the fusion gene, or the agent for measuring the transcription product of the fusion gene.

 It is still another object of the present invention to provide a colon cancer diagnostic kit comprising the composition for diagnosing colorectal cancer.

Yet another object of the present invention is to provide a method for providing information necessary for diagnosis of colon cancer, comprising the step of measuring the level of said NTRK1 fusion gene, its transcription product or its protein.

Yet another object of the present invention is to provide a method for providing information necessary for predicting colon cancer therapeutic prognosis, which comprises the step of measuring the level of said NTRK1 fusion gene, its transcription product or its protein.

It is still another object of the present invention to provide a method for screening a therapeutic agent for colorectal cancer comprising the step of measuring the expression level of the NTRK1 fusion gene.

It is still another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising an NTRK1 fusion protein inhibitor as an active ingredient.

As a result of intensive studies to achieve the above object, the inventors of the present invention have found that by sequencing the next generations of transcripts from 150 colon cancer clinical specimens and 50 normal colon tissues, TPM3-NTRK1 fusion The transcription product and the LMNA-NTRK1 fusion transcription product were detected. The TPM3-NTRK1 fusion gene was generated by the inversion between chromosome 1q21.3 and q23.1 regions, and the LMNA-NTRK1 fusion gene was generated by deletion between chromosome q22 and q23.1 regions.

The novel fusion gene and its encoded fusion transcript and fusion protein were detected in 3 out of 147 (2%) Korean colon cancer clinical tissues. From the above facts, it has been revealed that expression of fusion transcription products and fusion proteins in colon cancer is due to gene fusion.

Three colon cancer clinical tissues in which NTRK1 fusion transcripts were identified did not have somatic mutation of known colon cancer genes such as KRAS, NRAS and PIK3CA. In addition, when the above NTRK1 fusion gene was forcedly expressed in a non-tumorigenic NIH3T3 cell line, positive in vitro and in situ species were obtained. From these facts, it has been found that NTRK1 gene fusion is a driver oncogene in colorectal cancer.

The fusion protein of the present invention was confirmed in 29.2% of Korean patients with colorectal cancer and confirmed in 25.6% of Chinese patients with colorectal cancer. The fusion gene was observed in a high frequency in colon cancer of Northeast Asian people including Korean and Chinese It is the causative gene. In addition, the survival rate of a colon cancer patient having the fusion gene of the present invention or a fusion protein encoded by the fusion gene of the present invention is lower than the survival rate of a colorectal cancer patient having no fusion gene or the fusion protein of the present invention, And it is possible to use it as a diagnostic marker to predict the clinical prognosis of the patient.

It is believed that the gene fusion results in enhanced activation of the NTRK1 protein. In patients with colon cancer in which such activation is occurring, inhibitors of the NTRK1 protein are considered to be effective in treatment. Therefore, the present inventors have found that, in colorectal cancer, the efficacy of treatment with a drug can be predicted by using the fusion gene as a target, and furthermore, in the prediction, It has been found that effective treatment is possible when a drug is administered, and the present invention has been completed.

Accordingly, the present invention provides a method for diagnosing the clinical prognosis of patients with colorectal cancer based on the presence of the TPM3 gene or the fusion gene between the LMNA gene and the NTRK1 gene and the fusion transcription product and the fusion protein encoded thereby, The present invention relates to a method of determining the efficacy of treatment, and a method of treating colon cancer using the determination of efficacy, and more particularly, to provide the following invention.

In one aspect, the present invention provides a fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) with NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the fusion protein do.

Specifically, the present invention provides a fusion protein having the following structural formula.

A fragment containing the N-terminus of N- [LMNA (Lamin A)] - [a fragment containing the C-terminus of NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1]

A fragment containing the N-terminus of N- [TPM3 (Tropomyosin 3)] - [a fragment containing the C-terminus of NTRK1] -C.

The present invention also provides a fusion gene polynucleotide having the following structural formula.

5 '- [fragment containing the 5' end of the LMNA gene] - [fragment containing the 3 'end of the NTRK1 gene] -3'

5 '- [Fragment containing the 5' end of TPM3] - [Fragment containing the 3 'end of the NTRK1 gene] -3'.

That is, the present invention provides a fusion protein comprising a N-terminal fragment of LMNA or a fusion protein comprising a fragment containing N-terminal of TPM3 and a fragment containing C-terminal of NTRK1 and a fragment comprising the 5'- The fusion protein comprising a fragment comprising the 5 'end and a fragment comprising the 3' end of the NTRK1 gene are linked, wherein the LMNA, TPM3 or NTRK1 may be human. The fusion protein of the present invention may be mixed with an NTRK1 fusion protein, or alternatively, a LMNA-NTRK1 fusion protein or a TPM3-NTRK1 fusion protein.

As shown in the examples of the present invention, the present inventors first found an example of fusion of LMNA protein and NTRK1 protein in human colon cancer patients. The frequency and clinical prognosis of patients with colorectal cancer with fusion of the TPM3 gene or the LMNA gene with the NTRK1 gene and the fusion fusion product and fusion protein encoded thereby were first identified through a cohort analysis of large colorectal cancer patients (Table 1).

In the present invention, the TPM3 gene encoding the TPM3 protein may be derived from a human and is located on human chromosome 1 (q21.3), and the TPM3 protein encoded thereby is a polypeptide having a total amino acid length of 285 aa. The TPM3 protein or TPM3 protein fragment is the N-terminal fusion partner of the TPM3-NTRK1 fusion protein. For example, the TPM3 gene can be obtained from GenBank accession no. NM_152263, and the TPM3 protein may be a protein having an amino acid sequence encoded by NM_152263. In particular, the TPM3 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, and the TPM3 gene may be composed of the nucleic acid sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, the fragment containing the N-terminus of TPM3 has the amino acid sequence encoded by the nucleotide sequence from the eighth exon of NM_152263 (the nucleotide positions 154134289 to 154142876 based on the (-) strand on chromosome 1) , And one nucleotide (g) that does not form a codon at the 3 'end of exon 8 may exist (Table 2 and Table 3). In particular, the fragment containing the N-terminus of TPM3 may be composed of the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

The LMNA gene encoding the LMNA protein may be derived from a human and is located on human chromosome 1 (q22), and the encoded LMNA protein is a polypeptide having a total amino acid length of 664 aa. The LMNA protein or LMNA protein fragment is the N-terminal fusion partner of the LMNA-NTRK1 fusion protein. The LMNA gene is available from GenBank accession no. NM_170707, and the LMNA protein may be a protein having an amino acid sequence encoded by NM_170707. In particular, the LMNA protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27, and the LMNA gene may be composed of the nucleic acid sequence of SEQ ID NO: 28, but is not limited thereto.

In the present invention, the fragment containing the N-terminus of LMNA may have an amino acid sequence encoded by a nucleotide sequence from the sixth exon of NM_170707 (base position 156084504 to 156105740 based on the (+) strand on chromosome 1) , And one nucleotide (c) that does not form a codon at the 3 'end of exon 6 (Table 6 and Table 7). In particular, the fragment containing the N-terminus of the LMNA may be composed of the amino acid sequence of SEQ ID NO: 31, but is not limited thereto.

The NTRK1 gene encoding the NTRK1 protein can be derived from a human and is located on human chromosome 1 (q23.1), and the NTRK1 protein encoded thereby is a protein having a total amino acid length of 796 aa. The NTRK1 protein or NTRK1 protein fragment is the C-terminal fusion partner of the TPM3-NTRK1 fusion protein. For example, the NTRK1 gene can be obtained from GenBank accession no. NM_001012331, and the NTRK1 protein may be a protein having an amino acid sequence encoded by NM_001012331. In particular, the NTRK1 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, and the NTRK1 gene may be composed of the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO:

In the present invention, the fragment containing the C-terminus of NTRK1 has the nucleotide sequence of exon 9 of NM_001012331 (position 156830671 to 156844363 on the (+) strand of chromosome 1) or exon 11 (position 156830671 Or 156845312 nucleotide positions) or exon 12 (156830671 to 156845872 base positions relative to the (-) strand on chromosome 1) to the last exon. (Ac) of the 3 'end of exon 9, 2 nucleotides (gc) starting from the 3' end of exon 11, and 2 nucleotides starting from the 3 'end of exon 12 gt; may be in a form that does not form a codon, and when fused with a fragment of the TPM3 protein, the codon (gac or ggc or ggt) is linked to one nucleotide (g) To encode one amino acid (D or G). In particular, the fragment containing the C-terminus of NTRK1 may be composed of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, but is not limited thereto.

In this specification, break points (or fusion sites) of fragments of a fusion partner are described on the basis of the exon of the gene encoding them, and the N-terminus of the fragment (in the case of a C-terminal fusion partner) Or the intron site between the exon at the last (in the case of the N-terminal fusion partner) and the exon which is cleaved to be cleaved does not affect the amino acid sequence of the protein fragment to be encoded even when cleaved at any point in the C- The point to be cut may be any of the intron positions.

In the present invention, the LMNA-NTRK1 fusion gene may have the nucleotide sequence of SEQ ID NO: 33, and the LMNA-NTRK1 fusion protein may have the nucleotide sequence of SEQ ID NO: 34, but is not limited thereto. Also, the TPM3-NTRK1 fusion gene may have the nucleotide sequence of SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, and the TPM3-NTRK1 fusion protein may have the nucleotide sequence of SEQ ID NO: 24, SEQ ID NO: But is not limited thereto.

Unless otherwise stated in the present invention, all nucleotide sequences determined by sequencing cDNA, or DNA molecules derived from a gene, transcription product, or transcription product described herein, can be determined using an automated next generation nucleotide sequence sequencer or a lethal nucleotide sequence sequencer And all amino acid sequences encoded by the determined nucleotide sequence can be determined using an automated peptide sequencer. The nucleotide sequence determined by this automated approach may contain some errors compared to the actual sequence. For example, the nucleotide sequences determined by automated sequencing are typically at least about 90%, specifically at least about 95%, more specifically at least about 99%, more specifically at least about 99.9% sequence identity with the actual nucleotide sequence of the sequenced cDNA or DNA molecule And may have homology. May include one insert or deletion in the nucleotide determined relative to the actual sequence and such insertions or deletions may result in framehifts in translating the nucleotide sequence so that the encoded amino acid sequence is identical to the actual amino acid sequence and complete It can be different.

In another embodiment, the present invention provides a composition for diagnosing colorectal cancer comprising the fusion protein, the fusion gene, or an agent for measuring the transcription product of the fusion gene.

In the present invention, the fusion protein and the fusion gene are as described above.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the incidence of colon cancer.

In the present invention, the term " colon cancer " refers to a cancer arising from the mucosa, which is the innermost surface of the large intestine, and refers to rectal cancer, colon cancer and anal cancer.

In the present invention, the fusion protein and the fusion gene of LMNA or TPM3 and NTRK1 can be used as diagnostic markers for diagnosing colon cancer. In the present invention, the term " diagnostic markers, markers for diagnosis or diagnosis markers " Is a substance that can distinguish colon cancer cells from normal cells and includes polypeptides or nucleic acids (for example, mRNA) showing an increase pattern in cells having colon cancer as compared with normal cells, lipids, glycolipids, glycoproteins, (Monosaccharides, disaccharides, oligosaccharides, etc.), and the like. For the purpose of the present invention, the colon cancer diagnostic marker is a fusion gene of LMNA-NTRK1 (Lamin A-Neurotrophic Tyrosine Kinase, Receptor, Type 1) or TPM3-NTRK1 (Tropomyosin 3-Neurotrophic Tyrosine Kinase, Receptor, Type 1) Are genes whose expression is increased in tissues or cells.

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that have a high degree of validity with high diagnostic accuracy and high reliability to provide consistent results even in repeated measurements. The colon cancer diagnostic markers of the present invention show the same results in repeated experiments with genes whose expression always increases directly or indirectly with the onset of colorectal cancer and the difference in expression level is very large when compared with the control group, Are highly reliable markers with little chance of falling. Therefore, the diagnosis result based on the result of measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable. In this case, except for the genes expressing almost the same amount in the normal colon epithelial cells and the colon cancer cells, for example, the genes expressed in the normal tissues used as the control group, Can be selected.

In one embodiment of the present invention, the fusion gene of the TPM3 gene or the LMNA gene and the NTRK1 gene is a causative gene in colorectal cancer. By the above fusion, the expression of the NTRK1 protein is enhanced, and the NTRK1 protein is constantly activated And the diagnosis of poor prognosis caused by malignancy of colon cancer and the possibility of treatment with NTRK1 protein inhibitor are high.

Therefore, the NTRK1 fusion gene of the present invention or the fusion transcription product and fusion protein encoded thereby is found to be specifically detected or expressed in solid tumors, specifically in colorectal cancer patients. Therefore, the fusion gene or the fusion fusion product encoded therewith and fusion Proteins can be usefully used as markers for the prediction of prognosis of solid tumors, specifically colon cancer.

The level of expression of the fusion gene of the present invention in a biological sample can be confirmed by confirming the amount of the transcription product of the fusion gene, particularly mRNA and the amount of the fusion protein. Preferably, the agent for measuring the level of the fusion gene mRNA comprises a primer that specifically binds to the gene.

In the present invention, " measurement of mRNA expression level " is used to determine the presence and expression level of mRNA of colon cancer marker genes in a biological sample in order to diagnose colon cancer. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

In the present invention, the primer that specifically binds to the mRNA of the fusion gene is such that the primer that specifically binds to the mRNA of the fusion gene has a fusing point such that the region amplified by the primer includes the fusion point of two genes to be fused When the two fusion genes constituting the fusion gene are the TPM3 gene and the NTRK1 gene, a primer specific to the nucleotide sequence encoding the N-terminal of the TPM3 protein and a primer coding for the C-terminal of the NTRK1 A primer pair comprising a base sequence specific to a base sequence, and in particular, may be a primer pair consisting of a base sequence of SEQ ID NO: 35 and SEQ ID NO: 36, but is not limited thereto.

When the two fusion genes constituting the fusion gene are the LMNA gene and the NTRK1 gene, a primer specific to the nucleotide sequence encoding the N-terminus of the LMNA protein and a primer specific to the nucleotide sequence encoding the C-terminus of the NTRK1 And may be, in particular, a pair of primers consisting of the nucleotide sequence of SEQ ID NO: 37 and SEQ ID NO: 38, but is not limited thereto.

The term "primer " of the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand radiation. Primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences for each marker gene-specific primer. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides into homologues, and modifications between nucleotides, such as uncharged linkers such as methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

In the present invention, "agent for measuring a fusion gene" is a process for identifying the presence of a fusion gene of the present invention, which is a colon cancer marker gene, in a biological sample to diagnose colon cancer. For example, Or a probe or primer that binds to the target. In particular, probes that specifically bind to the fusion gene can use probes having the characteristics of Table 8, but are not limited thereto. In a specific example of the present invention, a probe of the NTRK1 Split FISH Probe (Cat # FS0024, Taiwan) marketed by Abnova was used.

In the present invention, " measurement of protein expression level " is a process for confirming the presence and expression level of a protein expressed from a fusion gene of the present invention, which is a colon cancer marker gene, in a biological sample in order to diagnose colon cancer, The amount of the protein can be confirmed using an antibody that specifically binds to the protein of the fusion gene.

In particular, the antibody that specifically binds to the fusion gene of the present invention may bind to the vicinity of the fusion site of LMNA or TPM3 with NTRK1 or the C-terminus of NTRK1, but is not limited thereto. Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, But are not limited to, immunoelectrophoresis, immunohistochemistry, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like. .

Preferably, the agent for measuring the level of the protein comprises an antibody specific for the protein.

In the present invention, " antibody " means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

As described above, since the novel colon cancer marker fusion protein has been identified in the present invention, the antibody can be easily produced using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for obtaining serum containing antibodies by injection of the colon cancer marker protein antigen into the animal and collection from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies may be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 624 Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

 In another embodiment of the present invention, there is provided a kit for colon cancer diagnosis comprising the composition for diagnosing colorectal cancer according to the present invention.

Specifically, the colorectal cancer diagnostic kit may be a reverse-electron polymerase-PCR (RT-PCR) kit, a DNA chip kit, or a protein chip kit. In particular, the colon cancer diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

In a specific embodiment, the diagnostic kit may be a diagnostic kit characterized by comprising essential elements necessary for performing a reverse transcription-polymerase reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits may be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC DEPC-water, sterile water, and the like.

In another embodiment, it may be a diagnostic kit, preferably comprising an essential element necessary for carrying out a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

Also preferably, it may be a diagnostic kit characterized by comprising essential elements necessary for performing a protein chip or conducting an ELISA. Protein chip kits or ELISA kits contain antibodies specific for the marker protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

In another aspect, the present invention provides a method for providing information necessary for diagnosing colorectal cancer, comprising the step of measuring the level of the NTRK1 fusion gene, its transcription product or its protein in order to provide information necessary for diagnosis of colon cancer to provide. Specifically, measuring the level of the NTRK1 fusion gene, its transcription product or its protein from a separate sample of individuals suspected of having colon cancer; And comparing the measured expression level of the fusion gene, its transcription product or its protein with a fusion gene of a normal control sample, its transcription product, or its protein expression level. to provide.

In the present invention, the NTRK1 fusion gene may be a polynucleotide constituting the 5 'end of the LMNA gene or the TPM3 gene; And a polynucleotide that constitutes the 3 ' end of the NTRK1 gene.

In the present invention, the term "individual" refers to an animal in which colon cancer can occur, so long as colon cancer is suspected. Examples of the disease that can obtain the information necessary for diagnosis through the above method include a TPM3 gene, a fusion gene of the LMNA gene and the NTRK1 gene, or a carcinoma whose expression of the NTRK1 gene is dependent on the fusion of the NTRK1 gene Especially colon cancer.

In the present invention, the term "sample of an individual" refers to a tissue in which the level of the mRNA of the LMNA-NTRK1 or TPM3-NTRK1 fusion gene, which is a colon cancer marker, or the protein level thereof differs in cells, tissues, organs, body fluids (DNA extracts, transcript extracts, cDNA preparations prepared from transcript extracts or aRNA preparations, etc.) obtained from these biological samples as well as digestive fluids, sputum, alveolar-bronchial washing liquids, urine, Extracts, and the like, but are not limited thereto. do. The sample may be subjected to a formalin fixing treatment, an alcohol fixing treatment, a freezing treatment, or a paraffin embedding treatment. In addition, the gene, transcription product, cDNA or protein can be prepared by selecting a well-known method appropriate for the type and condition of the sample.

Through the above detection methods, the expression level of the NTRK1 fusion gene, its transcription product or its protein in the normal control group can be compared with the expression level in suspected colon cancer, thereby diagnosing the actual colon cancer of the suspected colon cancer. That is, the expression level of the marker of the present invention was measured for cells suspected to be colon cancer, the expression level of the marker of the present invention was measured for normal cells, and then the expression level of the marker of the present invention was determined to be normal If cells are more expressed in a cell-like state than in a cell, it is possible to judge a cell that is supposed to be a colorectal cancer to be a colon cancer. That is, when the fusion gene, the transcription product thereof, or the protein expression level thereof measured from the separated sample of the subject suspected of having colorectal cancer is higher than the fusion gene of the normal control sample, the transcription product thereof or the protein expression thereof, Cancer. ≪ / RTI >

Analysis methods for measuring the level of mRNA or a protein of the fusion gene of LMNA-NTRK1 or TPM3-NTRK1 include Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, Tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, protein chips, and the like. Through the above analysis methods, it is possible to compare the amount of the antigen-antibody complex formed in the normal control group with the amount of the antigen-antibody complex formed in the suspected colon cancer, and the LMNA-NTRK1 or TPM3-NTRK1 fusion protein , And thus it is possible to diagnose the incidence of colon cancer in patients with suspected colon cancer.

In the present invention, the term antigen-antibody complex refers to a combination of a colon cancer marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label .

Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re.

Measurement of protein expression levels is preferably performed using an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. By checking the degree of complex formation between the colon cancer marker protein and the antibody, it can be confirmed whether or not the colon cancer has developed.

Further, preferably, Western blotting using one or more antibodies against the colon cancer marker is used. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the protein produced by expression of the gene can be confirmed by confirming the amount of the generated antigen-antibody complex using the labeled antibody, thereby confirming whether or not the colon cancer has occurred. The detection method comprises a method of examining the expression level of a marker gene in a control group and the expression level of a marker gene in a cell in which colon cancer has developed. The level of the peptide fragment may be expressed as the absolute (e.g., [mu] g / ml) or relative (e.g., relative intensity of the signal) difference of the marker protein.

Preferably, one or more antibodies against the colon cancer markers are arranged at predetermined positions on the substrate to use protein chips immobilized at a high density. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It is possible to confirm whether or not the colon cancer develops.

Specifically, in order to detect the fusion protein, fusion gene or transcription product in a clinical specimen of a suspected individual of a colorectal cancer of the present invention, it is preferable that (a) the fusion protein, the fusion gene encoding the fusion protein, (Add) and react with a substance that interacts with at least one selected from the group consisting of transcription products, And (b) detecting the reactant obtained through the process. The method may further include preparing a clinical sample before the step (a), and the step of preparing the clinical sample may include obtaining (separating) a clinical sample from a suspected colon cancer patient. In step (a), the interacting substance may be a compound, an antibody, an aptamer and a fusion gene or a transcription product (all or a part of the fusion protein, such as fusion Nucleic acid molecules (for example, primers, probes, aptamers, etc.) that bind to the target nucleic acid, and compounds. These interacting substances can be attached to, or used in conjunction with, one or more immunochemical labeling substances selected from the group consisting of free radicals, radioactive isotopes, fluorescent dyes, chromogenic substrates, enzymes, bacteriophages, . In the step (b), the reactant is a complex formed by interaction (binding) of at least one member selected from the group consisting of the fusion protein, the fusion gene, and the transcription product obtained in step (a) or complex. When the reactant is detected in the step of detecting the reactant, it can be determined that the fusion protein, the fusion gene or the transcription product is present.

A substance that interacts with a fusion gene encoding NTRK1 fusion protein or a corresponding transcription product may be a nucleic acid molecule capable of hybridizing with the fusion gene or transcription product. For example, the nucleic acid molecule may be an antisense oligonucleotide (e.g., siRNA, microRNA, etc.) that can specifically hybridize with the fusion gene in the clinical sample, the fusion site of the fusion gene, or the transcription product corresponding to the fusion gene or fusion site, , A probe (for example, 5 to 100 bp, 5 to 50 bp, 5 to 30 bp, or 5 to 25 bp), aptamer, and the like. For example, a fusion gene encoding the fusion protein, or a nucleic acid molecule capable of hybridizing with a transcription product molecule corresponding to the fusion gene, is fused to a continuous 50 to 250, particularly 100 to 200 bases containing a fusion site in the fusion gene 20 to 100 bp, or 25 to 50 bp in length, capable of hybridizing with 20 to 100, particularly 25 to 50, or complementary sequences adjacent to both ends of the polynucleotide fragment so as to amplify the polynucleotide fragment made, Lt; / RTI > The 'hybridization is possible' means that the base sequence is completely complementary to the base sequence to be detected, or has a complementary base sequence of 80% or more (for example, 80-100%), specifically 90% or more (for example, 90-100% Means that it can specifically bind to the gene or transcription product of interest.

In another example, a substance that specifically interacts with the NTRK1 fusion gene or a transcription product thereof may be one or more immunogens selected from the group consisting of free radicals, radioactive isotopes, fluorescent dyes, chromogenic substrates, enzymes, bacteriophages, A chemical labeling substance may be attached or used together with the labeling substance. For example, a probe for fluorescence in situ hybridization capable of hybridizing with each fusion gene and a pair of primers for a polymerase chain reaction can be used (Table 8 and Table 9).

In yet another example, detection of a NTRK1 fusion protein may be accomplished by contacting a substance that interacts with the NTRK1 protein that is expressed in a fusion protein or a colon cancer with the NTRK1 gene in a fusion-dependent manner, such as a specifically binding agent (e.g., an antibody, (E. G., Immunochromatography, immunohistochemistry (e. G., Immunohistochemistry) to detect the interaction, i. E. Complex formation, of the fusion protein with the material ), Enzyme linked immunosorbent assay, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, Western blotting blotting and flow cytometry cell sorting.

In another aspect, the present invention provides a method for providing information necessary for predicting colon cancer therapeutic prognosis, comprising the step of measuring the level of the NTRK1 fusion gene, its transcription product or its protein from a separate sample of colon cancer patients to provide.

Specifically, the present invention provides a method for diagnosing cancer, comprising measuring the level of a NTRK1 fusion gene, a transcription product thereof, or a protein thereof, from a separate sample of a colon cancer patient; And comparing the measured expression level of the fusion gene, its transcription product, or its protein with a fusion gene of a normal control sample, a transcription product thereof or a protein expression level thereof, to provide information necessary for predicting colorectal cancer treatment prognosis How to

The step of measuring the NTRK1 fusion gene, the transcription product thereof or the protein level thereof is almost the same as that described in the method for providing the information necessary for the diagnosis of colon cancer.

However, in the present invention, the sample to be measured is not a separate sample of suspected colon cancer but a separate sample of colon cancer patients. In the present invention, the normal control group is a normal group in which the NTRK1 fusion gene is not expressed, .

In the present invention, a method for providing information necessary for predicting colorectal cancer treatment prognosis is a method in which a NTRK1 fusion gene, a transcription product thereof, or a protein expression level thereof from a separate sample of colon cancer patients is compared with a fusion gene of a normal control sample, Protein expression level is predicted to be worse than that of the same or lower than that of the protein expression level. Also, comparing the NTRK1 fusion gene, its transcription product, or its protein expression level includes confirming the expression of the NTRK1 fusion gene, its transcription product or its protein. That is, it is predicted that patients with colorectal cancer expressed from NTRK1 fusion gene, its transcription product, or a patient with no colorectal cancer expressing the protein will be inferior in colorectal cancer treatment prognosis.

In addition, the present invention provides a method for providing information necessary for predicting the prognosis of colorectal cancer, which comprises detecting a fusion gene, a transcription product thereof, or a protein expression level thereof from a separate sample of a colon cancer patient with a fusion gene, a transcription product thereof, Is higher than its protein expression level, it may be characterized that it is predicted that the NTRK1 protein inhibitor is highly effective for colon cancer treatment.

Meanwhile, in the present invention, a method for providing information necessary for predicting colorectal cancer treatment prognosis is a method of treating NTRK1 fusion protein inhibitor to isolated cells of colon cancer patients and detecting the NTRK1 fusion gene, its transcription product or its protein And measuring the level.

In the present invention, the 'NTRK1 protein inhibitor' to be evaluated for the effectiveness of colorectal cancer treatment is not particularly limited as long as it is a substance capable of directly or indirectly inhibiting the function of the NTRK1 protein. A substance capable of inhibiting the function of the NTRK1 protein, specifically, tyrosine kinase, specifically, tyrosine kinase. Examples of known NTRK1 protein inhibitors applicable to the present invention include (5S, 6S, 8R) -6-hydroxy-6- (hydroxymethyl) -Trihydro-5H-16-oxa-4b, 8a, 14-triaza-5,8-methanoibenzo [b, h] cycloocta [jkl] cyclopenta [e] ) - a generic name for lestaurtinib: a compound targeting FLT3, JAK2, TRKA, TRKB and TRKC), N- [5 - ((2R) -2- methoxy- 3-yl] -4- (4-methylpiperazin-1-yl) benzamide (generic name: Tannu 8- ((4- (4-methylpiperazin-1-yl) -1H-pyrazol-3-yl) 4-yl) quinazoline-3-carboxamide (generic name: milciclib: CDK2, TRKA, TRKB, TRKC) Target compounds), RXDX-101 (code name: ROS1, ALK, TRKA) (Compounds targeting TRKA, TRKB, TRKC, ALK), PLX-7486 (a compound targeting CSF1R, TRKA, TRKB and TRKC), NMS-E628 (A compound targeting TRKA, TRKB, TRKC), a compound having the codename NMS-P626 (a compound targeting TRKA), a compound having a nucleotide sequence of SEQ ID NO: (Compounds targeting TRKA), and compounds having the code name ARRY-47 (compounds targeting TRKA, TRKB, and TRKC).

In a specific embodiment of the present invention, the 10-year survival period of 216 cohorts of Korean colon cancer patients was analyzed according to the expression of the protein encoded by the NTRK1 fusion gene. Sixty-three colon cancer patients expressing the NTRK1 fusion protein were analyzed for NTRK1 fusion protein The survival time was significantly shorter than that of the 153 colorectal cancer patients (Fig. 12). This indicates that the NTRK1 fusion gene and the fusion protein expressed therefrom in the colon cancer patients are inferior in colorectal cancer treatment prognosis.

In another aspect, the present invention provides a method for screening a therapeutic agent for colorectal cancer, comprising the step of measuring the expression level of the NTRK1 fusion gene. Specifically, the present invention provides a method for screening a candidate compound for treating colorectal cancer, ; And measuring the expression level of the NTRK1 fusion gene.

In the present invention, the NTRK1 fusion gene is as described above.

In the present invention, the measurement of the expression level of the NTRK1 fusion gene includes a method of measuring the level of the transcription product of the fusion gene and a method of measuring the level of the transcription product of the fusion gene and the protein thereof. The method of measuring the level is as described above.

The screening method of the therapeutic agent for colorectal cancer of the present invention may be judged to be a therapeutic agent for colorectal cancer when the expression level of the NTRK1 fusion gene is lowered, particularly by treatment with a candidate substance for the treatment of colorectal cancer.

Specifically, the method can be useful for screening a therapeutic agent for colorectal cancer by comparing the increase or decrease of the expression of the NTRK1 fusion gene in the presence or absence of a colorectal cancer treatment candidate substance. Substances that indirectly or directly reduce the concentration of the NTRK1 fusion gene (LMNA-NTRK1 or TPM3-NTRK1), or proteins expressed therefrom, can be selected as therapeutic agents for colon cancer. That is, the expression level of the marker NTRK1 fusion gene of the present invention in colon cancer cells is measured in the absence of a candidate for colorectal cancer treatment candidate, and the expression level of the marker NTRK1 fusion gene of the present invention is measured And comparing the quantities of the candidate agents with those of the candidate agent for colorectal cancer therapy, the agent can be predicted as a therapeutic agent for colorectal cancer when the level of expression of the marker of the present invention in the presence of the colorectal cancer therapeutic candidate substance is lower than the marker expression level in the absence of the colorectal cancer therapeutic candidate substance.

In another aspect, the present invention provides a substance screened with a therapeutic agent for colorectal cancer through the screening method of the colorectal cancer therapeutic agent.

In one specific embodiment of the present invention, the method for screening a therapeutic agent for colon cancer by inhibiting the function of the NTRK1 fusion protein was confirmed using a colon cancer cell line KM12. The multi-tyrosine kinase inhibitors Lestaurintinib and Krystotinib were purchased from Torquis Biosciences (UK). ARRY-470, a specific inhibitor of NTRK1 protein, was synthesized by LG Life Sciences and the compounds provided by Korea Advanced Institute of Science and Technology As a result of screening through the screening method of the present invention, Compound 8 (5f in the present invention) and Compound 27 (6s in the present invention) disclosed in Korean Patent Publication No. 10-2013-0106186 were screened. The screened compound 8 (5f) is a compound represented by the following formula (1) and the compound 27 (6s) is a compound represented by the following formula (2).

[Chemical Formula 1]

(2)

Each of the inhibitors was treated at a concentration of 0.64 nM at 10 μM for 4 days, and the degree of inhibition of cell proliferation was confirmed by using an ATP-Glo Bioluminometric Cell Viability Assay kit (Biotium Co., USA).

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of colon cancer, which comprises an NTRK1 fusion protein inhibitor as an active ingredient.

The NTRK1 fusion protein inhibitor may act to inhibit the activity of the fusion protein or the expression of the fusion gene. As described above, the NTRK1 fusion protein inhibitor is not particularly limited as long as it is a substance capable of directly or indirectly inhibiting the function of the NTRK1 protein, specifically the function of NTRK1 tyrosine kinase. As long as it can inhibit NTRK1 tyrosine kinase, it may be a substance that inhibits other tyrosine kinases. As a known RET tyrosine kinase inhibitor applicable to the present invention, for example, (5S, 6S, 8R) -6-hydroxy-6- (hydroxymethyl) 8,14,15-tetrahydro-5H-16-oxa-4b, 8a, 14-triaza-5,8-methanoibenzo [b, h] cycloocta [jkl] cyclopenta [e] (2R) -2-methoxy-l, 3-dihydro-lH-pyrazolo [ 3,4-c] pyrazol-3-yl] -4- (4-methylpiperazin-1-yl) benzamide (4-methylpiperazin-1-yl) - (4-methylpiperazin-1-yl) Yl) phenyl) amino) -4,5-dihydro-1H-pyrazolo [4,3-h] quinazoline-3-carboxamide (generic name: milciclib: CDK2, TRKA , TRKB, and TRKC), RXDX-101 TRKA, TRKB, TRKC, and ALK), PLX-7486 (codenamed CSF1R, TRKA, TRKB and TRKC as targets), TSR- (Compound targeting TRKA, TRKB and TRKC), code NMS-E628 (code name: a compound targeting ROS1, JAK2, ALK and TRKA) (A compound targeting TRKA), a compound with a code name of RXDX-102 (a compound targeting TRKA), a compound with a code name ARRY-47 (a compound targeting TRKA, TRKB, or TRKC) , Particularly ARRY-470, a compound represented by the following formula (1) or a compound represented by the following formula (2).

[Chemical Formula 1]

(2)

In addition, the NTRK1 fusion protein inhibitor may be an inhibitor of NTRK1 fusion gene expression, and may be selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the NTRK1 fusion gene. In addition, the NTRK1 fusion protein inhibitor may be an antibody that specifically binds to the NTRK1 fusion protein.

Such an antibody may be a polyclonal antibody, a monoclonal antibody, or a part thereof having an antigen binding property, and all immunoglobulin antibodies are included in the antibody of the present invention. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies, and in addition to the novel antibodies, antibodies that are already known in the art may also be included. In another aspect of the present invention, the antibody is an agent for inhibiting the expression of LMNA-NTRK1 or TPM3-NTRK1 fusion gene or a protein inhibitor expressed from the fusion gene as an active ingredient. As long as it has the characteristics of a binding that specifically recognizes the full length of two heavy chains and two light chains as well as functional fragments of the antibody molecule. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv.

The pharmaceutical composition for the treatment of colorectal cancer of the present invention is used in a method for removing NTRK1 fusion protein having an activity of increasing cancer migration in serum of colorectal cancer patients using an antibody specific for NTRK1 fusion protein, Colon cancer can be cured. The inventors of the present invention found that a compound showing an inhibitory effect of NTRK1 fusion protein through screening has an effect of inhibiting the proliferation of colon cancer cells, and thus it was confirmed that the NTRK1 fusion gene or a fusion protein expressed therefrom could be a target of colon cancer treatment Respectively.

Such a composition for treating colorectal cancer includes, but is not limited to, a substance capable of specifically binding to the NTRK1 fusion gene or a protein expressed therefrom to promote the anticancer activity. Examples thereof include proteins, aptamers, peptides, carbohydrates, Lt; / RTI >

The composition of the present invention may be administered together with a pharmaceutically acceptable carrier. In oral administration, a conjugate, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, In the case of injections, a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer and the like may be mixed. In the case of topical administration, a base, excipient, lubricant and preservative may be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, unit dosage ampoules or multiple dose forms may be prepared.

In another aspect, the present invention provides a method of preventing or treating colon cancer, which comprises administering to a subject a pharmaceutical composition for preventing or treating colon cancer comprising the NTRK1 fusion protein inhibitor as an active ingredient.

The NTRK1 fusion protein inhibitors, individuals and compositions of the present invention are as described above.

The composition of the present invention may be used alone, but it may be administered alone or in combination with radiation therapy or chemotherapy (including cell growth arrest or cytotoxic substance, antibiotic substance, alkylating agent, antimetabolite, hormone, An anti-EGFR agent, an anti-angiogenic agent, a paroxetine transferase inhibitor, an anti-angiogenesis inhibitor, a metallo-matrix protease inhibitor, a telomerase inhibitor, a tyrosine kinase inhibitor, an anti-growth factor receptor substance, , ras-raf signaling pathway pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin binders, topoisomerase I inhibitors, topoisomerase II inhibitors, etc.).

The term used in the present invention means administration of the composition of the present invention to a patient by any suitable method. The route of administration of the composition of the present invention is selected depending on the type of the inhibitor or the type of colon cancer, Lt; RTI ID = 0.0 > normal < / RTI > pathway. But are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, intraperitoneal administration, intraperitoneal administration, Do not.

The effective dose range of the composition of the present invention can be varied depending on the sex, body surface area, kind and severity of disease, age, sensitivity to the drug, administration route and discharge rate, administration time, treatment period, target cell, And may be readily determined by one of ordinary skill in the art.

The present invention provides diagnostic markers specific for colorectal cancer, thereby enabling accurate diagnosis of colorectal cancer, predicting clinical prognosis through stratification of colorectal cancer patients, predicting the efficacy of treatment for colorectal cancer, In particular, it becomes possible to predict the efficacy of treatment of colon cancer with the NTRK1 protein inhibitor. This makes it possible to limit the administration of a drug to a colon cancer patient who is thought to be ineffective in administering the drug, thus making it possible to realize an efficient and safe customized colon cancer treatment.

Fig. 1 shows the results of sequencing analysis of the NTRK1 gene by NTRK1 gene fusion in 150 colon cancer clinical tissues and 3 colon cancer clinical tissues derived from 50 identical patients from the same patient using HiSeq 2000 of Illumina Co., This figure shows that the expression of the transcription product is increased and the data analysis result.
FIG. 2 is a figure showing the result of analysis and data showing that NTRK1 is fused with LMNA or TPM3 by analyzing the base sequence by Sanger sequencing of the fusion fusion product encoded from the NTRK1 fusion gene.
FIG. 3 is a graph showing the expression of NTRK1 protein having a NTRK1 fusion gene and analyzing expression of NTRK1 protein in colon cancer and normal colon tissue pairs in which the NTRK1 fusion gene has been identified at the transcript level.
FIG. 4 shows (a) colon cancer in which the NTRK1 fusion gene has been identified at the transcription product and protein level through a sequencing analysis of the next generation transcript; (b) Fusion of NTRK1 gene in normal colon tissue pairs is analyzed at the DNA level by fluorescence in situ hybridization, which shows the fusion of NTRK1 gene specific for colon cancer. The yellow arrow represents the NTRK1 gene isolated by gene fusion. The reference represents 5 micrometers.
5 is a diagram showing a single fusion site of the LMNA gene and the NTRK1 gene and three fusion sites of the TPM3 gene and the NTRK1 gene.
6 is a diagram showing the structure of a fusion protein encoded by a fusion gene of LMNA gene and NTRK1 gene, and a fusion gene of TPM3 gene and NTRK1 gene.
FIG. 7 is a graph showing the results of analysis of somatic cell mutations of a cancer gene-specific gene and a repressor gene in 147 colon cancer clinical tissues through a sequencing analysis of a next-generation transcript. The colon cancer clinical tissue having the NTRK1 fusion gene was analyzed for colon cancer- It is confirmed that there is no somatic mutation.
FIG. 8 shows the expression of the NTRK1 fusion gene inducing the in vitro tumorigenicity of the NIH3T3 cell line by forcibly expressing the LMNA-NTRK1 and TPM3-NTRK1 fusion genes in the non-tumorigenic NIH3T3 cell line and evaluating the colony formation assay Fig. An empty vector without the NTRK1 fusion gene was forcibly expressed as a negative control, and a KM12 colon cancer cell line with a TPM3-NTRK1 fusion gene as a positive control was used.
FIG. 9 is a graph showing the expression of NTRK1 fusion gene and the expression of NTRK1 fusion gene after inoculation into the dorsal subcutaneously of nude mice lacking immune function by forcibly expressing the LMNA-NTRK1 and TPM3-NTRK1 fusion genes in the non-tumorigenic NIH3T3 cell line Lt; RTI ID = 0.0 > NIH3T3 < / RTI > cell line. A KM12 colon cancer cell line with a TPM3-NTRK1 fusion gene was used as a positive control.
Figure 10 shows the expression of the protein encoded by the NTRK1 gene in tissue microarrays derived from 216 Korean colon cancer patients cohort and tissue microarrays derived from 472 Chinese colon cancer patient cohorts, And the expression of NTRK1 protein was detected in 25.6% of the Chinese colon cancer cohort.
FIG. 11 is a graph showing the fluorescence direct binding method that the fusion frequency of the NTRK1 gene was increased as the expression level of the NTRK1 protein in the clinical tissue derived from the cohort of the colon cancer patient who was confirmed to have been expressed by the NTRK1 gene was confirmed. The yellow arrow represents the NTRK1 gene isolated by gene fusion. The reference represents 10 micrometers.
FIG. 12 is a graph showing the survival time of 216 Korean colon cancer patient cohorts according to the expression of the protein encoded by the NTRK1 gene. In 63 colon cancer patients expressing NTRK1 protein, 153 colon cancer patients without NTRK1 protein expression The survival time was significantly shorter than that of the patients.
Fig. 13 is a graph showing the effect of the inhibition of NTRK1 protein against lestaurtinib and NTRK1 protein in the KM12 colon cancer cell line with the TPM3-NTRK1 fusion gene and the inhibitory effect of crizotinib and NTRK1 (5f) and Compound 27 (5s) and ARRY-470 disclosed in Korean Patent Laid-Open No. 10-2013-0106186, which is a protein-specific inhibitor, were applied at different concentrations to further reduce KM12 cell proliferation in NTRK1-specific inhibitors Fig.
FIG. 14 is a graph showing that the intermediate level of 118.5 million aligned leads were produced through sequencing analysis of the next generation transcript, and the level at which one base out of 100 bases could have an error was 94.3%.
FIG. 15 shows three outlier clinical tissues obtained by analyzing whole clinical tissues using PCA (Principle Component Analysis) based on the expression amount of 18,725 genes recognized in 80% or more of clinical tissues.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One : LMNA  or TPM3 - NTRK1  The fusion gene and the encoded Warrior mountain Water and fusion protein

In the present invention, an example of the fusion of the LMNA protein and the NTRK1 protein in colon cancer has been disclosed for the first time in the present invention through the following examples. Also, the frequency and clinical prognosis of patients with colorectal cancer having a fusion of TPM3 gene or LMNA gene with NTRK1 gene and a fusion fusion product and fusion protein encoded thereby are first identified in the present invention through a large-scale colorectal cancer patient cohort analysis.

1-1. NTRK1 Wow The fusion protein  Gene

First, the NTRK1 fusion gene specifically present in the colon cancer tissue identified in the present invention is summarized in Table 1 below.

Colon cancer-specific NTRK1 fusion gene Donor gene domain The acceptor gene domain Distance (Mb) TPM3 1q21.3 NTRK1 1q23.1 2.6 LMNA 1q22 NTRK1 1q23.1 0.6

The NTRK1 fusion gene of the present invention is cleaved and fused at the break point (fusion site) of the protein fragments compiled in Table 1 above. Is cleaved at any point in the intron region between the exon contained at the end of the fragment (in the case of a C-terminal fusion partner) or the C-terminal (in the case of an N-terminal fusion partner) and the truncated exon of the fragment The amino acid sequence of the protein fragment is not affected, so that the point at which the protein is actually cleaved may be any of the intron positions.

1-2. TPM3 - NTRK1  Fusion genes and TPM3 - NTRK1 Fusion protein

In the present invention, the TPM3 gene encoding the TPM3 protein is a human TPM3 gene. The TPM3 protein encoded by the chromosome 1 (q21.3) is a protein having a total amino acid length of 285 aa. The TPM3 protein or TPM3 protein fragment is the N-terminal fusion partner of the TPM3-NTRK1 fusion protein.

In the present invention, the TPM3 protein uses human-derived TPM3, and the NTRK1 gene encoding the same is located on human chromosome 1 (q21.3). The TPM3 protein encoded by this is a protein with a total amino acid length of 285 aa. The TPM3 protein or TPM3 protein fragment is the N-terminal fusion partner of the TPM3-NTRK1 fusion protein.

In the present invention, the above-mentioned GenBank accession no. The TPM3 gene having the nucleotide sequence of NM_152263 was used.

The fragment of the TPM3 protein used was one having an amino acid sequence encoded by a nucleotide sequence from the eighth exon of NM_152263 (positions 154134289 to 154142876 bases on the (-) strand on chromosome 1). There was one nucleotide (g) that did not form a codon at the 3 'end of the exon 8 encoding the TPM3 protein used in the present invention. The genes encoding the TPM3 protein fragments and the TPM3 proteins encoded therefrom are summarized in Tables 2 and 3 below.

A gene encoding a fragment of the TPM3 protein TPM3 gene
Accession No. TPM3 protein coding region
(CDS) TPM3 protein fragment Encoding site:
Exon basis TPM3 protein fragment Encoding site:
cDNA standard Chromosome image
Cutting position 5 'end cutting position
Base sequence NM_152263 118 to 975 (858 bp)
(SEQ ID NO: 1) Exon 1 to exon 8 118 to 891 + 1 nt (g) (775 bp) (SEQ ID NO: 2) chr1: 154142876 5'-ATT GAT GAC CTG GAA g-3 '
(SEQ ID NO: 3)

Fragment of TPM3 protein Overall length of TPM3 protein
(Accession No.) TPM3 protein fragment site N-terminal direction cleavage position
Amino acid sequence 285aa (NP_689476)
(SEQ ID NO: 4) 1 to 258aa + 1nt (g) (amino acid sequence: SEQ ID NO: 5) N-IDDLE + 1nt (g) (amino acid sequence: SEQ ID NO: 6)

In the present invention, the NTRK1 protein uses human-derived NTRK1, and the NTRK1 gene encoding the same is located on human chromosome 1 (q23.1). The NTRK1 protein encoded thereby is a protein with a total amino acid length of 796 aa. The NTRK1 protein or NTRK1 protein fragment is the C-terminal fusion partner of the TPM3-NTRK1 fusion protein.

In the present invention, GenBank accession no. The NTRK1 gene having the nucleotide sequence of NM_001012331 was used.

The fragment of the NTRK1 protein is located at the base position of exon 9 of NM_001012331 (position 156830671-156844363 base on the (+) strand of chromosome 1) or exon 11 (position 156830671-156845312 base of the (-) strand on chromosome 1) And those having an amino acid sequence encoded by a nucleotide sequence from exon 12 (position 156830671 to 156845872 base on the (-) strand on chromosome 1) to the last exon were used. (Ac) of the 3 'end of exon 9, 2 nucleotides (gc) starting from the 3' end of exon 11, and 2 nucleotides starting from the 3 'end of exon 12 gt) contained sequences that did not form codons. The sequences that do not form the codon are fused to a fragment of the TPM3 protein to form a codon (gac or ggc or ggt) linked to one nucleotide (g) additionally contained in the fragment of the TPM3 protein, (D or G). The gene encoding the NTRK1 protein fragment and the NTRK1 protein expressed therefrom are summarized in Tables 4 and 5 below.

A gene encoding a fragment of the NTRK1 protein NTRK1 gene
(Accession No.) The coding region (CDS) of the NTRK1 protein NTRK1 protein fragment Encoding site:
Exon basis NTRK1 protein fragment Encoding site:
cDNA standard Chromosomal cleavage site 3 'terminal direction cleavage site sequence NM_002529 52 to 2447 (2396 bp)
(SEQ ID NO: 7) Exon 9 to exon 17 2nt (ac) + 1236 to 2447 (1214 bp) (SEQ ID NO: 8) chr1: 156844363 5'-ac ACT AAC AGC ACA TCT-3 '(SEQ ID NO: 9) NM_002529 52 to 2447 (2396 bp)
(SEQ ID NO: 7) Exon 11 to exon 17 2nt (gc) + 1393 to 2447 (1055 bp) (SEQ ID NO: 10) chr1: 156845312 5'-gc CCG GCT GTG CTG GCT-3 '(SEQ ID NO: 11) NM_002529 52 to 2447 (2396 bp)
(SEQ ID NO: 7) Exon 12 to exon 17 2nt (gt) + 1540 to 2447 (908 bp) (SEQ ID NO: 12) chr1: 156845872 Gt; GTT CAC < / RTI > CAC ATC AAG -3 '(SEQ ID NO: 13)

Fragment of NTRK1 protein The total length of the NTRK1 protein (Accession No.) NTRK1 protein fragment site C-terminal direction cleavage site Amino acid sequence 796aa (NP_002520)
(SEQ ID NO: 14) 2nt (ac) + 400-796 (amino acid sequence: SEQ ID NO: 15) 2nt (ac) + TNSTS-C (amino acid sequence: SEQ ID NO: 16) 796aa (NP_002520)
(SEQ ID NO: 14) 2nt (gc) + 453-796 (amino acid sequence: SEQ ID NO: 17) 2nt (gc) + PAVLA-C (amino acid sequence: SEQ ID NO: 18) 796aa (NP_002520)
(SEQ ID NO: 14) 2nt (gt) + 502-796 (amino acid sequence: SEQ ID NO: 19) 2nt (gt) + VHHIK-C (amino acid sequence: SEQ ID NO: 20)

A fusion gene (TPM3-NTRK1 fusion gene) encoding the TPM3-NTRK1 fusion protein in which the NTM1 protein or a fragment thereof is fused with the TPM3 protein or a fragment thereof is a nucleotide encoding a TPM3 protein or a fragment thereof at the 5 ' And a nucleotide molecule encoding the NTRK1 protein or fragment thereof as described above at the 3 ' end.

As shown in the above table, in the present invention, the nucleotide sequence of exon 1 to exon 8 of NM_152263 and the exon 9 or exon 11 or exon 12 to exon 17 of NM_002529 on the 3 'end of the 5' end of the TPM3-NTRK1 fusion gene A fusion gene to which the nucleotide sequence was linked was prepared. Specifically, in the present invention, the nucleotide sequence from the 118th to 892nd nucleotides of the NM_152263 (SEQ ID NO: 2) and the nucleotide sequence from the 1234th or 1391th or the 1338th to 2447th nucleotides of the NM_002529 (SEQ ID NO: 21: a base sequence comprising SEQ ID NO: 3 and SEQ ID NO: 9; SEQ ID NO: 22: a fusion protein comprising SEQ ID NO: 3 and SEQ ID NO: 11) Nucleotide sequence; SEQ ID NO: 23: a nucleotide sequence comprising SEQ ID NO: 3 and SEQ ID NO: 13).

In addition, the TPM3-NTRK1 fusion protein encoded from the TPM3-NTRK1 fusion gene prepared in the present invention has an amino acid sequence (SEQ ID NO: 5) of NP_689476 at the N terminus and an amino acid sequence at the 400th or (SEQ ID NO: 24: an amino acid sequence comprising SEQ ID NO: 6 and SEQ ID NO: 16; SEQ ID NO: 25, SEQ ID NO: : An amino acid sequence comprising SEQ ID NO: 6 and SEQ ID NO: 18; SEQ ID NO: 26: an amino acid sequence including SEQ ID NO: 6 and SEQ ID NO: 20).

1-3. NMNA - NTRK1  Fusion genes and NMNA - NTRK1 Fusion protein

The LMNA gene encoding the LMNA protein, which is a representative example of the present invention, uses a human-derived LMNA gene and is located on chromosome 1 (q22) of human, and the LMNA protein encoded thereby is a protein having a total amino acid length of 664 aa. The LMNA protein or LMNA protein fragment is the N-terminal fusion partner of the LMNA-NTRK1 fusion protein.

In the present invention, GenBank accession no. The TPM3 gene having the nucleotide sequence of NM_170707 was used.

The fragments of the LMNA protein were those having an amino acid sequence encoded by a nucleotide sequence from the sixth exon of NM_170707 (positions 156084504 to 156105740 bases on the (+) strand of chromosome 1). There was one nucleotide (c) that did not form a codon at the 3 'end of exon 6 that encodes the LMNA protein used in the present invention. The genes encoding the fragments of the LMNA protein and the LMNA proteins encoded therefrom are summarized in Tables 6 and 7 below.

A gene encoding a fragment of LMNA protein LMNA gene
(Accession No.) The LMNA protein coding region (CDS) LMNA protein fragment Encoding site: exon standard LMNA protein fragment Encoding site: cDNA standard Chromosomal cleavage site 5 'terminal direction cleavage site sequence NM_170707 250 to 2244 (1995 bp) (SEQ ID NO: 27) Exon 1 to exon 6 250 to 1233 + 1 nt (c) (985 bp) (SEQ ID NO: 28) chr1: 156105740 5'-GAG GAC TCA CTG GCC c-3 '(SEQ ID NO: 29)

Fragment of LMNA protein The total length of the LMNA protein (Accession No.) LMNA protein fragment site N-terminal direction cleavage site Amino acid sequence 664aa (NP_733821) (SEQ ID NO: 30) 1 to 328aa + 1nt (c) (amino acid sequence: SEQ ID NO: 31) N-EDSLA + 1nt (g) (amino acid sequence: SEQ ID NO: 32)

In the present invention, a fusion gene (LMNA-NTRK1 fusion gene) encoding the LMNA-NTRK1 fusion protein in which the NTRK1 protein or a fragment thereof is fused with the LMNA protein or a fragment thereof is obtained by introducing the LMNA protein or a fragment thereof at the 5 ' Encoding nucleotide molecule and a nucleotide molecule encoding the NTRK1 protein or fragment thereof described in Example 1-2 above at the 3 'end.

Specifically, in the present invention, the LMNA-NTRK1 fusion gene to which the nucleotide sequence from exon 1 to exon 6 of NM_170707 and the nucleotide sequence from exon 11 to exon 17 of NM_002529 were linked to the 3 'end of the 5' end was prepared. Specifically, in the present invention, a nucleotide sequence from the 250th to 1234th nucleotides of NM_170707 (SEQ ID NO: 28) and a nucleotide sequence from the 1391th to 2447th nucleotides of the NM_002529 (SEQ ID NO: 10) -NTRK1 fusion gene (SEQ ID NO: 33: a nucleotide sequence comprising SEQ ID NO: 29 and SEQ ID NO: 11).

In addition, the LMNA-NTRK1 fusion protein encoded by the LMNA-NTRK1 fusion gene prepared in the present invention has the N-terminal amino acid sequence (SEQ ID NO: 31) of the LMNA protein and the NTRK1 protein (SEQ ID NO: 34: amino acid sequence comprising SEQ ID NO: 32 and SEQ ID NO: 18) was linked to the amino acid sequence of SEQ ID NO: 18 from SEQ ID NO:

Example  2: NTRK1  The fusion gene or its Warrior product  Measuring means

In the present invention, in order to analyze the frequency and clinical prognosis of patients with colorectal cancer having the NTRK1 fusion gene (fusion gene of TPM3 gene or LMNA gene and NTRK1 gene) and the fusion fusion product and fusion protein encoded thereby, NTRK1 fusion gene or its transcription product. However, in the present invention, a probe for fluorescence in situ hybridization (FISH) (NTRK1 Split FISH Probe, Cat # FS0024, Abnova, Taiwan) capable of hybridizing with the NTRK1 fusion gene was used and NTRK1 fusion transcription A hybridizable mid-chain hybrid application primer to the product was prepared.

Probes for fluorescence direct conjugation that can hybridize to the NTRK1 fusion gene Junction Length Labeling substance The 5 'end of SEQ ID NO: 7
(chr1: 156390000 to 156814000) Approximately 420 kb TexRed The 3 ' end of SEQ ID NO: 7
(chr1: 156851000 to 157630000) Approximately 780 kb FITC

Synthesized conjugate application primers hybridizable to the NTRK1 fusion transcription product Fusion gene Fusion site Forward Primer Sequence Reverse Primer Sequence TPM3-NTRK1 SEQ ID NO: 21;
SEQ ID NO: 22;
SEQ ID NO: 23 5'-AAG AAG ATA AAT ATG AGG-3 '
(SEQ ID NO: 35) 5'-CCG TGC CGC ATA TAC TCA AA-3 '
(SEQ ID NO: 36) LMNA-NTRK1 SEQ ID NO: 33 5'-CCA GGT GGA GCA GTA TAA GAA G -3 '
(SEQ ID NO: 37) 5'-TGT GGG TTC TCG ATG ATG TG -3 '
(Sequence defense 38)

In addition, the detection of the NTRK1 fusion protein may be accomplished by using a substance that specifically interacts with the NTRK1 protein, such as an antibody, an aptamer or a compound, that expresses in a fusion protein or a colon cancer in a manner dependent on the fusion of the NTRK1 gene, Such as immunochromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and the like, to detect the interaction of the fusion protein with the material (e.g., antibody or aptamer) An enzyme-linked immunosorbent assay, a radioimmunoassay, an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay, a western blotting, Can be detected by flow cytometry cell sorting or the like. For example, antibodies for immunohistochemistry for detecting NTRK1 protein are shown in Table 10 below. In the present invention, an antibody for immunohistochemistry (Anti-NTRK1 Antibody EP1058Y (Cat # TA301109, OriGene, USA)) for detecting NTRK1 protein was used.

Antibodies for immunohistochemistry that interact with NTRK1 fusion proteins antigen Action site Negative contrast Positive contrast Other applications The 791st tyrosine of the NTRK1 protein amino acid sequence and its surrounding peptides The kinase domain C-terminus of the NTRK1 protein Lymphocytes Ganglion cells Western blotting, fluorescence immunoassay, flow cytometry

Example  3: Experiment using clinical tissue

The fresh frozen clinical tissue used in the present invention was collected from patients with colorectal cancer who underwent surgical resection at Pusan National University Hospital and Chonnam National University Hospital from 2008 to 2012, It was preserved at Chonnam National University Hospital Hwasoon branch of Hwasun city and sold. Colon cancer tissues were selected according to the guidelines of the International Cancer Genome Consortium (ICGC). For example, clinical tissues used only clinical tissues composed of more than 60% cancer cells and less than 20% necrotic cells or normal cells after pathological diagnosis. Colon cancer clinical specimens collected from colon cancer patients with family history of colon cancer were excluded. Finally, 150 colon cancer tissues and 50 normal colon tissues were used.

Example  4: Brown rice  Instability analysis microsatellite instability 분석 : MSI )

The whole DNA was extracted from formalin-fixed paraffin-embedded tissues after formalin fixation in the colon cancer clinical tissue of Example 3, and then analyzed using Bethesda marker (BAT26, D5S346, BAT25, D17S250, D2S123) Body instability.

Specifically, the whole DNA was extracted using a QIAmp DNA FFPE Tissue kit (KIAJEN, Germany). DNA purity and concentration were determined by ND-100 spectrophotometer (NanoDrop Technologies, USA). Microsatellite instability was determined according to the amplification product length and signal intensity obtained after multiplex polymerisation. When chain polymerization failed due to incompleteness of the extracted DNA, brown solid anxiety was judged by considering the total number of somatic mutations of the major gene including DNA polymerase epsilon (POLE).

Example  5: Next generation Transcript  Sequence analysis ( RNA - seq )

Total RNA was extracted with RNAiso Plus (Takada Bioscience, Japan), pre-treated with DNase I (Kiagen), and purified with RNeasy Mini kit (KIAJEN). RNA integrity was analyzed by Bioanalyer (Agilent, USA) and RNA-derived RNA from colon cancer clinical tissue with an RNA Integrity Number (RIN) of 6 or greater was applied to the RNA-seq. The library for RNA-seq was prepared with a TruSeq RNA sample preparation kit (Illuminas, USA). For example, mRNA was recovered with magnetic beads attached with poly-T oligo and then fragmented by acoustic shearing. Primary cDNA was prepared from the fragmented mRNA using reverse transcriptase and random hexamer and secondary cDNA was prepared using DNA polymerase I and RNase H. Secondary cDNA was ligated to the adapter and then subjected to a chain polymerization to complete a cDNA library. The cDNA library was sequenced using HiSeq 2000 (Illuminosa) and produced approximately 100 million paired-end reads of 101 bp in length.

Example  6: Sequence analysis

The paired end leads produced by the sequencing analysis of the next generation transcript in Example 5 were obtained from National Center for Biotechnology Information (NCBI, USA) under conditions permitting 5% discrepancy using GSNAP and TopHat. Lt; / RTI > (hg 19), which is provided by < RTI ID = 0.0 > Fairend Reid also mRNA splicing by aligning to a human reference transcript consisting of 161,250 mRNAs extracted from the public database (36,742 mRNA from the RefSeq database, 73,671 mRNA from the USCS database, and 161,214 mRNA from the Ensembl database) To minimize misalignment.

Example  7: Fusion gene analysis

The in-frame fusion gene was searched through the GFP algorithm and cross-validated with the deFuse and FusionMap algorithms. For example, fusion genes through GFP were searched under the following conditions.

(1) The discordant read pairs and the exon-fusion breakpoints are ligated to different donor genes and acceptor genes constituting the fusion body, spanning reads, if any.

(2) Donor and housekeeping genes If the strand orientation of the DNA strand describes gene fusion through chromosomal translocation or inversion or deletion, accept it.

(3) When the homology of the nucleotide sequence is high (E value <0.01).

(4) Excludes cases where the distance is less than 10bp for illogical lead pairs.

The fusion gene was confirmed by the following conditions.

(1) Accept fusion genes found in two algorithms: GFP or deFuse or FusionMap.

(2) Accept if there are more than 10 lead pairs aligned in the donor gene and in the storage gene.

(3) Accept if more than 10 leads are aligned at the fusion site.

(4) Accepts when the donor gene and the storage gene are present in the same chromosome and the intergenic distance is 100 Kb or more.

(5) The out-frame fusion gene is excluded.

(6) The exon of the stored gene existing after the fusion site is accepted when compared with the exon of the unfused storage gene.

Example  8: abnormal expression gene ( differentially expressed gene : DEG ) analysis

The overexpressed genes were analyzed using the GenePattern software of the Broad Institute of the United States. The next generation transcript was sequenced and TopHat aligned leads were assembled into a transcript using Cufflinks. The number of lined leads for each gene was standardized by FPKM (Fragments Per Kilobase of Exon per Million) method and expressed as the expression level of each gene. Based on the expression of 18,725 genes with over 0 FPKM in over 80% of clinical tissues, all clinical tissues were analyzed by PCA (Principle Component Analysis) to remove 3 outlier clinical tissues (6 pairs). Through the PCA analysis, 18,725 genes obtained from 147 colon cancer clinical specimens and 47 normal clinical specimens were subjected to abnormal expression gene analysis. Specific abnormal expression genes in colon cancer tissues were retrieved by pairwise 2-sided t-test (p <0.05) and Benjamini-Hochart multiple comparison correction (FDR <0.05).

Example  9: somatic cell mutation ( non - synonymous somatic mutation ) analysis

Single nucleotide variances (SNVs) were searched by next-generation transcript sequencing analysis by aligning leads produced by GSNAP to human reference transcripts. Single base mutations were detected under the following conditions.

(1) Accept if the number of leads that are position-specifically aligned is two or more.

(2) Accept if the average base quality at this location is 20 or more.

(3) Accept if the frequency of the allele in the location is 3% or more.

(4) If the number of leads aligned at the above position is 10 or more, accept.

The annotation of the gene was referred to RefSeq and the germline variants were removed under the following conditions.

(1) Excludes cases in which the frequency of minor alleles in all clinical tissues is greater than 1%, as compared to dbSNP137.

(2) Excludes germ cell mutations observed in normal individuals compared to 59 Korean reference genomes.

(3) The germ cell mutations observed in 47 normal colon tissues used in the present invention are excluded.

Considering that the incidence of somatic mutations higher than expected in the colon cancer clinical tissue without normal colon tissue pairs was observed, somatic cell mutations were searched based on negative selection.

Example  10: Reverse transcription polymerization reverse transcriptase - 중합체 chain  reaction: RT - PCR )and Fishing Sequencing ( Sanger sequencing )

NTRK1 fusion transcripts were confirmed by reverse transcription polymerase chain reaction and seaweed sequencing. The conditions of the chain polymerization reaction are summarized in Table 11 below.

Sequence polymerization conditions for confirmation of NTRK1 fusion transcript Donor gene Storage gene primer Chain polymerization reaction conditions Chain polymerization product TPM3 (exon 8) NTRK1 (exon 9 or exon 11 or exon 12) SEQ ID NO: 35 and SEQ ID NO: 36 Denaturation: 94 degrees 30 seconds
Annealing: 55.7 degrees 30 seconds
Extension
72 degrees 30 seconds
30 times in total 406bp
553bp
712bp LMNA (exon 6) NTRK1 (exon 11) SEQ ID NO: 37 and SEQ ID NO: 38 Denatured: 94 degrees 30 seconds
Combination: 54 degrees 30 seconds
Height: 72 degrees 30 seconds
30 times in total 354bp

The product of the chain polymerization was inserted into the TOPO TA vector (Life Technologies, USA), and sequenced by a bioluminescence method.

Example  11: Fluorescence direct bonding ( 여광체 in situ hybridization : FISH )

The NTRK1 fusion gene was identified using a split fluorescent direct probe (Avnova, Taiwan) that binds to the 5 'and 3' ends of the NTRK1 gene, respectively. For example, cut sections of paraffin-embedded colon cancer and normal clinical tissue after formalin fixation were de-paraffinized and denatured at 75 degrees after treatment with protease. The sections of the denatured clinical tissue were incubated for 17 hours in ThermoBrite (Abbott, USA) with the probes listed in Table 8. The cut sections of the clinical tissues were cleaned and stained with DAPI (Abbott), and the distance between TexRed and FITC signals was observed.

Example  12: Immunohistochemical analysis immunohistochemistry ) And clinicopathological ( clinicopathological ) analysis

The expression of NTRK1 fusion protein was immunohistologically confirmed using the NTRK1 protein specific antibody (Orijin, USA) as shown in Table 10, in view of the fact that NTRK1 fusion protein expresses dependent on NTRK1 gene fusion. Brain ganglion cells and lymphocytes were used as positive and negative controls for immunohistochemical analysis of NTRK1 fusion protein expression, respectively.

The expression frequency of the NTRK1 fusion protein was analyzed immunohistochemically using a tissue microarray constructed in 216 Korean colon cancer patient cohorts and 472 Chinese colon cancer patient cohorts (Biomax, USA). In immunohistochemical analysis of NTRK1 fusion protein expression, Korean patients with colorectal cancer were stratified into NTRK1 fusion protein positive patients and negative patients. In the stratified group, colon cancer location, invasion depth, perineural invasion ), Lymphovascular emboli, lymphnode metastasis, microscopic instability, and 10-year survival rates.

Example  13: NIH3T3  In the cell line NTRK1  Expression of fusion gene

The cDNAs of the TPM3, LMNA and NTRK1 genes were purchased from Origin. The fusion products of LMNA (exon 6) -NTRK1 (exon 11) and TPM3 (exon 8) -NTRK1 (exon 9 or exon 11 or exon 12) were prepared by overlap extension chain polymerization. The resulting fusion transcription product was cloned into pcDNA3.1 (Life Technologies) equipped with IRES-GFP using a Cold Fusion Cloning kit (System Biosciences, USA). The cloned fusion gene was confirmed by bioassay sequencing and the fusion gene expression vector for TPM3-NTRK1 and LMNA-NTRK1 fusion was completed. The NIH3T3 cell line was purchased from the Korean Cell Line Bank. Fusion transcription product expression vector was introduced into NIH3T3 cell line using FUGENE 6 (Roche, Germany). The transformed NIH3T3 cell line was selected using G418 (RIKE TECHNOLOGY INC.) And completed by flow cytometry analysis using GFP protein expression.

Example  14: NTRK1  Of the fusion gene Bred  analysis

Tumorigenicity of the TPM3-NTRK1 or LMNA-NTRK1 fusion gene was confirmed by the ability of the transformed NIH3T3 cell line to in vitro cell mass-forming ability and the tumorigenicity. In vitro cell mass formation ability was examined by counting 1,200 transfected NIH3T3 cells in 0.3% top agarose and culturing for 3 weeks on 0.5% lower agarose. The NIH3T3 cell line into which the empty vector without the fusion gene was introduced and the KM12 colon cancer cell line with the TPM3-NTRK1 fusion gene were used as negative and positive control, respectively. The formed cell mass was stained with 0.05% crystal violet and then reconstituted with an optical microscope. In vivo tumorigenicity was observed after inoculation of a total of one million transgenic NIH3T3 cells or KM12 cells in dorsal subcutaneously of nude mice lacking immunity. Five nude mice were used for each experimental group. After cell inoculation, the size of the tumor was measured for a total of 32 days after cell inoculation at 3 days intervals when a measurable tumor was formed.

Example  15: Drug Screening

The screening method for the treatment of colorectal cancer by inhibiting the function of the NTRK1 fusion protein was confirmed using a KM12 cell line. The multi-tyrosine kinase inhibitors Lestaurintinib and Krystotinib were purchased from Torquis Biosciences (UK). ARRY-470, a specific inhibitor of NTRK1 protein, was synthesized by LG Life Sciences and the compounds provided by Korea Advanced Institute of Science and Technology As a result of screening through the screening method of the present invention, Compound 8 (5f in the present invention) and Compound 27 (6s in the present invention) disclosed in Japanese Patent Laid-Open No. 10-2013-0106186 were screened. The screened compound 8 (5f) is a compound represented by the following formula (1) and the compound 27 (6s) is a compound represented by the following formula (2).

[Chemical Formula 1]

(2)

Each of the inhibitors was treated at a concentration of 0.64 nM at 10 μM for 4 days, and the degree of inhibition of cell proliferation was confirmed by using an ATP-Glo Bioluminometric Cell Viability Assay kit (Biotium Co., USA).

As a result of screening for a drug capable of treating colon cancer by inhibiting the activity of the NTRK1 fusion gene or the NTRK1 fusion protein encoded by the NTRK1 fusion gene, lestauritinib, which has activity on the NTRK1 protein among the multiple tyrosine kinase inhibitors, Cell growth was inhibited by 50% and ARRY-470, a selective inhibitor of the NTRK1 protein, inhibited KM12 cell growth by 50% at about 3.2 nM. On the other hand, chrysotanib, which has low activity on the NTRK1 protein, inhibited the growth of KM12 cells by 50% at a concentration of about 184.8 nM. Compound 8 (5f in the present invention) and Compound 27 (6s in the present invention) disclosed in Patent Publication No. 10-2013-0106186 having high selectivity for the NTRK1 protein exhibited a relatively low KM12 cell at 544.2 nM and 929.1 nM due to low cell permeability (Fig. 13).

Example  16: observed in Korean colon cancer clinical tissue Inflammation  Fusion gene

The next generation transcript sequencing analysis yielded a median 118.5 million aligned leads, and 94.3% of the 100 bases were found to be error-prone (see FIG. 14). PCA analysis showed that three clinical tissues were abnormal and three pairs of colon cancer and normal colon tissue containing abnormal tissue were excluded from the present invention (see FIG. 15). Thus, 147 cases of colon cancer and 47 cases of normal colon cancer were used in the present invention. Fusion genetic analysis using GFP or deFuse or FusionMap algorithms identified nine infiltrating fusion genes (see Table 12).

Inflammation fusion gene observed in Korean colon cancer clinical tissue Sample ID Stage Discordant paired-end reads spanning reads Distance
(bp) Donor gene
(Donor gene) FPKM Location Awarded gene
(Acceptor) FPKM Location LGP088T Ⅲ 27 153 2620933 TPM3 59 chr1: 154142876 NTRK1 31 chr1: 156844363 LGC012T Ⅲ 25 132 2620933 TPM3 94 chr1: 154142876 NTRK1 20 chr1: 156845312 TPM3 94 chr1: 154142876 NTRK1 20 chr1: 156845872 LGC026T Ⅱ 11 15 675664 LMNA 145 chr1: 156105740 NTRK1 32 chr1: 156845312 LGC007T Ⅲ 21 197 771740 PTPRK 17 chr6: 128841404 RSPO3 31 chr6: 127469793 LGC054T Ⅲ 36 140 771740 PTPRK 15 chr6: 128505577 RSPO3 37 chr6: 127469793 LGC030T Ⅲ 56 374 2667510 NAGLU 255 chr17: 40693224 IKZF3 55 chr17: 37922746 LGP047T Ⅱ 45 206 1020112 GTF3A 641 chr13: 27999075 CDK8 93 chr13: 26923209 LGP024T Ⅱ 24 98 55463 RAD51AP1 7 chr12: 4662255 AKAP3 18 chr12: 4737971 LGP031T Ⅱ 17 53 28829615 RASA1 11 chr5: 86564807 LOC644100 19 chr5: 115394422

The fusion genes identified include tyrosine dephosphorylase, receptor type, K (tyrosine phosphatase, receptor type, K: PTPRK) gene and R-spondin 3 (RSPO3) (1.4%), and a fusion gene between the N-acetylglucosaminidase (alpha: NAGLU) gene and the IKAROS family zinc finger 3 (IKZF3) gene One case of colon cancer was confirmed in clinical tissue (0.7%), and the fusion gene between the general transcription factor IIIA (GTF3A) gene and the cyclin-dependent kinase 8 (CDK8) (0.7%), a fusion gene between the RAD51 associated protein 1 (RAD51AP1) gene and the A kinase anchor protein 3 (AKAP3) gene was found in one case of colorectal cancer In clinical tissue (RAS p21 protein activator 1: RASA1) and the ADP-ribosylation factor-like 14 effector protein-like (LOC644100) gene The gene was identified in one case of colorectal cancer (0.7%), and the gene for lamin A / C (LMNA) or tropomyosin 3 (TPM3) and neurotrophic tyrosine kinase, receptor type 1 (neurotrophic tyrosine kinase, receptor, type 1: NTRK1) gene was identified in three colon cancer clinical tissues (2.0%). Among the fusion genes, the NTRK1 gene encodes the TrkA protein immobilized on the cell membrane. TPM3-NTRK1 fusion gene has been reported in colorectal cancer and thyroid papillary cancer. Myosin phosphatase Rho interaction protein interacting protein (MPRIP) gene or a fusion gene between CD74 gene and NTRK1 gene has been reported. In Spitzoid melanoma, a fusion gene between TP53 gene or LMNA gene and NTRK1 gene has been reported. In gallbladder cancer, RAB A fusion gene between protein 1 (RAB GTPase activating protein 1-like: RABGAP1L) gene and NTRK1 gene has been reported. In the polymorphic orphan species, fusion of neurofascin (NFASC) gene or brevica Genes were reported. However, the frequency and clinical significance of NTRK1 fusion gene in colorectal cancer has not been clarified.

Example  17: NTRK1  gene, NTRK1  Fusion gene and its protein analysis

Expression of the NTRK1 gene exon was observed only in the colon cancer clinical tissue in which the NTRK1 fusion gene was identified (see FIG. 1). The fusion gene of NTRK1 was analyzed using reverse transcription polymerase chain reaction and seaweed sequencing. As a result, the TPM3-NTRK1 fusion gene was cleaved at the position of chr1: 154142876 among the TMP3 gene to preserve SEQ ID NO: 2, specifically SEQ ID NO: Terminus and conserved in SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 in the NTRK1 gene at the position of chr1: 156844363 or chr1: 156845312 or chr1: 156845872 and specifically SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: It was confirmed that Fusion 13 was conserved in the 3 'terminal direction. The LMNA-NTRK1 gene was cleaved at the chr1: 156105740 position of the LMNA gene to preserve the meristematic 28, specifically, the sequence SEQ ID NO: 29 was conserved at the 5'-terminal direction, and the truncated at the chr1: 156845312 position of the NTRK1 gene, (SEQ ID NO: 11) was conserved in the 3 'terminal direction (see FIG. 2).

Immunohistochemical analysis of antibody against NTRK1 protein, specifically NTRK1 protein encoded by NTRK1, specifically antibody binding to the C-terminal region of TrkA protein revealed that TRKA expression in clinical colon cancer tissues confirmed NTRK1 fusion transcription product (See FIG. 3). The NTRK1 fusion gene was reaffirmed by fluorescence direct conjugation assay for the NTRK1 gene. The 5 'end of the NTRK1 gene was conjugated with a TexRed-labeled probe, and the 3' end of the NTRK1 gene was ligated with a FITC-labeled probe. As a result, NTRK1 fusion gene product and fusion protein Red and green probe signals were identified (see FIG. 4), and red and green probe signals were combined in normal colon tissue (see FIG. 5). As a result of the NTRK1 fusion gene and the fusion protein, it was confirmed that the NTRK1 fusion protein has a well-conserved tyrosine kinase domain in the NTRK1 protein (see FIG. 6).

Example  18: NTRK1  Of the fusion gene Bred  Confirm

Tumorigenicity of the NTRK1 fusion gene has been reported in papillary thyroid carcinoma and lung cancer. However, the tumorigenicity of the NTRK1 fusion gene in colorectal cancer has not been adequately verified. In silico and in vitro and in vivo evaluations were carried out to confirm the heterogeneity of TPM3-NTRK1 fusion gene and LMNA-NTRK1 fusion gene. Based on the results of the somatic cell mutation analysis, the tumorigenicity of the NTRK1 fusion gene was analyzed by the in silico method. As a result, somatic mutation of the typical colon cancer gene was not observed in the colon cancer clinical tissue in which the NTRK1 fusion gene was identified (see FIG. 7). It was confirmed that the NTRK1 fusion gene acts as a driver oncogene in colon cancer. NIH3T3 cells transformed through forced expression of the NTRK1 fusion gene formed cell masses similar to those of the KM12 colon cancer cell line in vitro. In contrast, the NIH3T3 cell line transfected with a blank vector lacking the NTRK1 fusion gene did not form cell mass (see FIG. 8). The NIH3T3 cell line transfected with transformed NIH3T3 cells in nude mice lacking immunity was able to measure from day 18 after cell inoculation and the KM12 colon cancer cell line at day 32 after cell inoculation Developed to a similar level of tumor (see FIG. 9).

Example  19: NTRK1  Protein expression and NTRK1  Correlation of fusion genes

In view of the fact that the NTRK1 protein expresses dependent on the NTRK1 gene fusion, a tissue microarray constructed from 216 Korean colon cancer patient cohorts and 472 Chinese colorectal cancer patient cohorts, and NTRK1 protein, specifically TrkA As a result of observing the frequency of the NTRK1 fusion gene using an antibody that binds to the C-terminal region of the protein, NTRK1 protein expression was confirmed in 29.2% of colon cancer tissue derived from Korean patients with colon cancer, and 25.6% It was confirmed that NTRK1 protein is expressed in colon cancer-derived clinical tissues derived from cancer patients (see FIG. 10). To confirm the correlation between NTRK1 protein expression and NTRK1 fusion gene, fluorescent direct conjugation of NTRK1 gene was performed on the clinical tissues of Korean patients with colorectal cancer whose NTRK1 protein expression was confirmed from a Korean colorectal cancer patient cohort. The frequency of isolated NTRK1 fluorescent direct junction signals was observed in clinical tissues in which the expression of NTRK1 protein was confirmed (p < 0.0192) (see Fig. 11 and Table 13). Thus, the NTRK1 fusion gene was confirmed to be a part of NTRK1 protein expression, specifically 42.9% of the causative genes.

Relationship between NTRK1 protein expression and NTRK1 fusion gene Sample ID Cytoplasmic TrkA expression NTRK1 fusion P value IHC FISH Colon_50_FISH01 Strong Frequently detected 0.0192 Colon_50_FISH02 Strong Not detected Colon_50_FISH03 Strong Not available Colon_50_FISH04 Strong Frequently detected Colon_50_FISH05 Strong Not detected Colon_50_FISH06 Moderate Frequently detected Colon_50_FISH07 Moderate Not detected Colon_50_FISH08 Mild Not detected Colon_50_FISH09 Mild Not detected Colon_50_FISH10 Mild Not detected Colon_50_FISH11 Negative Not detected Colon_50_FISH12 Negative Not detected Colon_50_FISH13 Negative Not detected Colon_50_FISH14 Negative Not detected Colon_50_FISH15 Negative Not detected

Example  20: NTRK1 Fusion protein  Survival rate of colorectal cancer patients

A cohort analysis of 216 colon cancer patients who were followed up among the colon cancer patients who received the clinical sample was conducted. Forty - two patients (24.1%) died during the follow - up period in the follow - up 10 year survival rate for 216 Korean patients with colorectal cancer.

First, according to the results of clinical sample analysis, 216 patients with colorectal cancer were stratified into NTRK1 fusion protein positive patients and NTRK1 fusion protein negative patients according to the expression of NTRK1 protein. Clinical pathologic analysis of patients with stratified colon cancer revealed that the NTRK1 fusion gene or the NTRK1 fusion protein encoded in the colon cancer patient group had colon cancer in the left colon (p = 0.0504), high colon cancer invasion (p (P = 0.0437), positive neural invasion or lymphovascular embolus was observed (p = 0.0429), lymph node metastasis was high (p = 0.015), and brownish anomalies were stable (p = 0.0024) .

Clinical pathologic features of colorectal cancer patients (Korean) cohort N Cytoplasmic TrkA P value (-) (+) Age (years) 216 70.35 70.22 0.4675 Size (cm) 216 5.199 5.548 0.2959 gender 0.3351 Male 118 85 33 Female 98 68 30 location 0.0504 Right colon 87 67 20 Left colon 129 86 43 Histological type 0.0761 well 16 13 3 Moderate 178 120 58 Poor 10 10 0 Mucinous 12 10 2 Invasion depth
(T1-2 vs. T3) 0.0437 T1 12 10 2 T2 23 19 4 T3 181 124 57 Perineural invasion
(PNI) 0.1315 Negative 149 109 40 Positive 67 44 23 Lymphovascular emboli (LVE) 0.2229 Negative 142 103 39 Positive 74 50 24 PNI / LVE 0.0429 Negative 119 90 29 Positive 97 63 34 Lymphnode metastasis
(N0 vs N1-2) 0.015 N0 124 95 29 N1a 40 23 17 N1b 32 22 10 N2a 13 9 4 N2b 7 4 3 Microsatellite status 0.0024 MSS 180 119 61 MSI-L 6 5 One MSI-H 30 29 One

The 10-year survival rate of the colon cancer group was significantly lower than that of the NTRK1 fusion protein-free colon cancer patients (p = 0.0411, risk = 0.5346, 95% confidence interval = 0.2548 to 0.9722) (see FIG. 12).

Example  21: Conclusion

As described above, according to the present invention, the prognosis of patients with colorectal cancer can be evaluated by detecting transcription products and proteins of the NTRK1 gene, which are dependent on the fusion of the TPM3-NTRK1 fusion gene or the fusion fusion protein and the fusion protein or NTRK1 gene encoded thereby And to predict the efficacy of treatment of colon cancer with the NTRK1 protein inhibitor.

Inhibitors that have an inhibitory effect on the NTRK1 protein have already been introduced in clinical studies for the treatment of various carcinomas. For example, lestaurtinib is studying the therapeutic effects of prostate cancer, neuroblastoma, and myeloid leukemia, while danucer tips are studying the therapeutic effects of prostate cancer, non-small cell lung cancer and chronic myelogenous leukemia, RXDX-102 is a treatment for other carcinomas, while TSR-011 for non-small cell lung cancer, NMS-P626 for colorectal cancer, and RXDX-102 for other carcinomas The effects are under study.

Thus, the fusion of the NTRK1 gene or the expression of the transcription product and protein of the NTRK1 gene dependent thereon occurs in multiple carcinomas. The NTRK1 fusion gene is diverging from the mutation of colon cancer genes such as KRAS, NRAS and PIK3CA. Thus, the NTRK1 fusion gene can be a target of a conventional NTRK1 protein inhibitor, specifically a tyrosine kinase inhibitor having an inhibitory effect on NTRK1 tyrosine kinase. The presence of the NTRK1 fusion gene is also recognized in Chinese colorectal cancer patients, including Koreans. Therefore, the method of the present invention is very useful in increasing the efficiency of colorectal cancer treatment in Korean and Chinese patients with colorectal cancer.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> LG Life Sciences Ltd. <120> Novel NTRK1 fusion gene as colon cancer marker and the uses          the <130> KPA140037-KR-P1 <150> KR 10-2014-0004491 <151> 2014-01-14 <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 858 <212> DNA <213> TPM3 CDS <400> 1 atgatggagg ccatcaagaa aaagatgcag atgctgaagt tagacaagga gaatgctctg 60 gatcgggcag agcaagctga agctgagcag aagcaggcag aagaaagaag taaacagctg 120 gaggatgagc tggcagccat gcagaagaag ctgaaaggga cagaggatga gctggacaag 180 tattctgaag ctttgaagga tgcccaggag aagctggaac tggcagagaa gaaggctgct 240 gatgctgagg ctgaggtggc ctccttgaac cgtaggatcc agctggttga agaagagctg 300 gaccgtgctc aggagcgcct ggccactgcc ctgcaaaagc tggaagaagc tgaaaaagct 360 gctgatgaga gtgagagagg tatgaaggtt attgaaaacc gggccttaaa agatgaagaa 420 aagatggaac tccaggaaat ccaactcaaa gaagctaagc acattgcaga agaggcagat 480 aggaagtatg aagaggtggc tcgtaagttg gtgatcattg aaggagactt ggaacgcaca 540 gaggaacgag ctgagctggc agagtctaag tgttctgagc tggaggagga gctgaagaat 600 gtcaccaaca acctcaagtc tcttgaggct caggcggaga agtactctca aaaagaagat 660 aaatatgagg aagaaatcaa gattcttact gataaactca aggaggcaga gacccgtgct 720 gagtttgctg agagatcggt agccaagctg gaaaagacaa ttgatgacct ggaagatgag 780 ctctatgccc agaaactgaa gtacaaggcc attagcgagg agctggacca cgccctcaat 840 gacatgacct ctatataa 858 <210> 2 <211> 775 <212> DNA <213> TPM3 fragment cDNA <400> 2 atgatggagg ccatcaagaa aaagatgcag atgctgaagt tagacaagga gaatgctctg 60 gatcgggcag agcaagctga agctgagcag aagcaggcag aagaaagaag taaacagctg 120 gaggatgagc tggcagccat gcagaagaag ctgaaaggga cagaggatga gctggacaag 180 tattctgaag ctttgaagga tgcccaggag aagctggaac tggcagagaa gaaggctgct 240 gatgctgagg ctgaggtggc ctccttgaac cgtaggatcc agctggttga agaagagctg 300 gaccgtgctc aggagcgcct ggccactgcc ctgcaaaagc tggaagaagc tgaaaaagct 360 gctgatgaga gtgagagagg tatgaaggtt attgaaaacc gggccttaaa agatgaagaa 420 aagatggaac tccaggaaat ccaactcaaa gaagctaagc acattgcaga agaggcagat 480 aggaagtatg aagaggtggc tcgtaagttg gtgatcattg aaggagactt ggaacgcaca 540 gaggaacgag ctgagctggc agagtctaag tgttctgagc tggaggagga gctgaagaat 600 gtcaccaaca acctcaagtc tcttgaggct caggcggaga agtactctca aaaagaagat 660 aaatatgagg aagaaatcaa gattcttact gataaactca aggaggcaga gacccgtgct 720 gagtttgctg agagatcggt agccaagctg gaaaagacaa ttgatgacct ggaag 775 <210> 3 <211> 16 <212> DNA <213> TPM3 5'terminal cleavage site seq <400> 3 attgatgacc tggaag 16 <210> 4 <211> 285 <212> PRT <213> TPM3 full length <400> 4 Met Met Glu Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys   1 5 10 15 Glu Asn Ala Leu Asp Arg Ala Glu Gln Ala Glu Ala Glu Gln Lys Gln              20 25 30 Ala Glu Glu Arg Ser Lys Gln Leu Glu Asp Glu Leu Ala Ala Met Gln          35 40 45 Lys Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Ala      50 55 60 Leu Lys Asp Ala Gln Glu Lys Leu Glu Leu Ala Glu Lys Lys Ala Ala  65 70 75 80 Asp Ala Glu Ala Glu Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val                  85 90 95 Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln             100 105 110 Lys Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met         115 120 125 Lys Val Ile Glu Asn Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu     130 135 140 Gln Glu Ile Gln Leu Lys Glu Ala Lys His Ile Ala Glu Glu Ala Asp 145 150 155 160 Arg Lys Tyr Glu Glu Val Ala Arg Lys Leu Val Ile Ile Glu Gly Asp                 165 170 175 Leu Glu Arg Thr Glu Glu Arg Ala Glu Leu Ala Glu Ser Lys Cys Ser             180 185 190 Glu Leu Glu Glu Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Ser Leu         195 200 205 Glu Ala Gln Ala Glu Lys Tyr Ser Gln Lys Glu Asp Lys Tyr Glu Glu     210 215 220 Glu Ile Lys Ile Leu Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala 225 230 235 240 Glu Phe Ala Glu Arg Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp                 245 250 255 Leu Glu Asp Glu Leu Tyr Ala Gln Lys Leu Lys Tyr Lys Ala Ile Ser             260 265 270 Glu Glu Leu Asp His Ala Leu Asn Asp Met Thr Ser Ile         275 280 285 <210> 5 <211> 258 <212> PRT <213> TPM3 fragment <400> 5 Met Met Glu Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys   1 5 10 15 Glu Asn Ala Leu Asp Arg Ala Glu Gln Ala Glu Ala Glu Gln Lys Gln              20 25 30 Ala Glu Glu Arg Ser Lys Gln Leu Glu Asp Glu Leu Ala Ala Met Gln          35 40 45 Lys Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Ala      50 55 60 Leu Lys Asp Ala Gln Glu Lys Leu Glu Leu Ala Glu Lys Lys Ala Ala  65 70 75 80 Asp Ala Glu Ala Glu Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val                  85 90 95 Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln             100 105 110 Lys Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met         115 120 125 Lys Val Ile Glu Asn Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu     130 135 140 Gln Glu Ile Gln Leu Lys Glu Ala Lys His Ile Ala Glu Glu Ala Asp 145 150 155 160 Arg Lys Tyr Glu Glu Val Ala Arg Lys Leu Val Ile Ile Glu Gly Asp                 165 170 175 Leu Glu Arg Thr Glu Glu Arg Ala Glu Leu Ala Glu Ser Lys Cys Ser             180 185 190 Glu Leu Glu Glu Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Ser Leu         195 200 205 Glu Ala Gln Ala Glu Lys Tyr Ser Gln Lys Glu Asp Lys Tyr Glu Glu     210 215 220 Glu Ile Lys Ile Leu Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala 225 230 235 240 Glu Phe Ala Glu Arg Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp                 245 250 255 Leu Glu         <210> 6 <211> 5 <212> PRT <213> TPM3 N terminus cleavage site seq <400> 6 Ile Asp Asp Leu Glu   1 5 <210> 7 <211> 2391 <212> DNA <213> NTRK1 CDS <400> 7 atgctgcgag gcggacggcg cgggcagctt ggctggcaca gctgggctgc ggggccgggc 60 agcctgctgg cttggctgat actggcatct gcgggcgccg caccctgccc cgatgcctgc 120 tgcccccacg gctcctcggg actgcgatgc acccgggatg gggccctgga tagcctccac 180 cacctgcccg gcgcagagaa cctgactgag ctctacatcg agaaccagca gcatctgcag 240 catctggagc tccgtgatct gaggggcctg ggggagctga gaaacctcac catcgtgaag 300 agtggtctcc gtttcgtggc gccagatgcc ttccatttca ctcctcggct cagtcgcctg 360 aatctctcct tcaacgctct ggagtctctc tcctggaaaa ctgtgcaggg cctctcctta 420 caggaactgg tcctgtcggg gaaccctctg cactgttctt gtgccctgcg ctggctacag 480 cgctgggagg aggagggact gggcggagtg cctgaacaga agctgcagtg tcatgggcaa 540 gggcccctgg cccacatgcc caatgccagc tgtggtgtgc ccacgctgaa ggtccaggtg 600 cccaatgcct cggtggatgt gggggacgac gtgctgctgc ggtgccaggt ggaggggcgg 660 ggcctggagc aggccggctg gatcctcaca gagctggagc agtcagccac ggtgatgaaa 720 tctgggggtc tgccatccct ggggctgacc ctggccaatg tcaccagtga cctcaacagg 780 aagaacgtga cgtgctgggc agagaacgat gtgggccggg cagaggtctc tgttcaggtc 840 aacgtctcct tcccggccag tgtgcagctg cacacggcgg tggagatgca ccactggtgc 900 atccccttct ctgtggatgg gcagccggca ccgtctctgc gctggctctt caatggctcc 960 gtgctcaatg agaccagctt catcttcact gagttcctgg agccggcagc caatgagacc 1020 gtgcggcacg ggtgtctgcg cctcaaccag cccacccacg tcaacaacgg caactacacg 1080 ctgctggctg ccaacccctt cggccaggcc tccgcctcca tcatggctgc cttcatggac 1140 aaccctttcg agttcaaccc cgaggacccc atccctgtct ccttctcgcc ggtggacact 1200 aacagcacat ctggagaccc ggtggagaag aaggacgaaa caccttttgg ggtctcggtg 1260 gctgtgggcc tggccgtctt tgcctgcctc ttcctttcta cgctgctcct tgtgctcaac 1320 aaatgtggac ggagaaacaa gtttgggatc aaccgcccgg ctgtgctggc tccagaggat 1380 gggctggcca tgtccctgca tttcatgaca ttgggtggca gctccctgtc ccccaccgag 1440 ggcaaaggct ctgggctcca aggccacatc atcgagaacc cacaatactt cagtgatgcc 1500 tgtgttcacc acatcaagcg ccgggacatc gtgctcaagt gggagctggg ggagggcgcc 1560 tttgggaagg tcttccttgc tgagtgccac aacctcctgc ctgagcagga caagatgctg 1620 gtggctgtca aggcactgaa ggaggcgtcc gagagtgctc ggcaggactt ccagcgtgag 1680 gctgagctgc tcaccatgct gcagcaccag cacatcgtgc gcttcttcgg cgtctgcacc 1740 gagggccgcc ccctgctcat ggtctttgag tatatgcggc acggggacct caaccgcttc 1800 ctccgatccc atggacctga tgccaagctg ctggctggtg gggaggatgt ggctccaggc 1860 cccctgggtc tggggcagct gctggccgtg gctagccagg tcgctgcggg gatggtgtac 1920 ctggcgggtc tgcattttgt gcaccgggac ctggccacac gcaactgtct agtgggccag 1980 ggactggtgg tcaagattgg tgattttggc atgagcaggg atatctacag caccgactat 2040 taccgtgtgg gaggccgcac catgctgccc attcgctgga tgccgcccga gagcatcctg 2100 taccgtaagt tcaccaccga gagcgacgtg tggagcttcg gcgtggtgct ctgggagatc 2160 ttcacctacg gcaagcagcc ctggtaccag ctctccaaca cggaggcaat cgactgcatc 2220 acgcagggac gtgagttgga gcggccacgt gcctgcccac cagaggtcta cgccatcatg 2280 cggggctgct ggcagcggga gccccagcaa cgccacagca tcaaggatgt gcacgcccgg 2340 ctgcaagccc tggcccaggc acctcctgtc tacctggatg tcctgggcta g 2391 <210> 8 <211> 1196 <212> DNA <213> NTRK1 fragment cDNA <400> 8 acactaacag cacatctgga gacccggtgg agaagaagga cgaaacacct tttggggtct 60 cggtggctgt gggcctggcc gtctttgcct gcctcttcct ttctacgctg ctccttgtgc 120 tcaacaaatg tggacggaga aacaagtttg ggatcaaccg cccggctgtg ctggctccag 180 aggatgggct ggccatgtcc ctgcatttca tgacattggg tggcagctcc ctgtccccca 240 ccgagggcaa aggctctggg ctccaaggcc acatcatcga gaacccacaa tacttcagtg 300 atgcctgtgt tcaccacatc aagcgccggg acatcgtgct caagtgggag ctgggggagg 360 gcgcctttgg gaaggtcttc cttgctgagt gccacaacct cctgcctgag caggacaaga 420 tgctggtggc tgtcaaggca ctgaaggagg cgtccgagag tgctcggcag gacttccagc 480 gtgaggctga gctgctcacc atgctgcagc accagcacat cgtgcgcttc ttcggcgtct 540 gcaccgaggg ccgccccctg ctcatggtct ttgagtatat gcggcacggg gacctcaacc 600 gcttcctccg atcccatgga cctgatgcca agctgctggc tggtggggag gatgtggctc 660 caggccccct gggtctgggg cagctgctgg ccgtggctag ccaggtcgct gcggggatgg 720 tgtacctggc gggtctgcat tttgtgcacc gggacctggc cacacgcaac tgtctagtgg 780 gccagggact ggtggtcaag attggtgatt ttggcatgag cagggatatc tacagcaccg 840 actattaccg tgtgggaggc cgcaccatgc tgcccattcg ctggatgccg cccgagagca 900 tcctgtaccg taagttcacc accgagagcg acgtgtggag cttcggcgtg gtgctctggg 960 agatcttcac ctacggcaag cagccctggt accagctctc caacacggag gcaatcgact 1020 gcatcacgca gggacgtgag ttggagcggc cacgtgcctg cccaccagag gtctacgcca 1080 tcatgcgggg ctgctggcag cgggagcccc agcaacgcca cagcatcaag gatgtgcacg 1140 cccggctgca agccctggcc caggcacctc ctgtctacct ggatgtcctg ggctag 1196 <210> 9 <211> 17 <212> DNA <213> NTRK1 3 'terminal cleavage site seq <400> 9 acactaacag cacatct 17 <210> 10 <211> 1037 <212> DNA <213> NTRK1 fragment cDNA <400> 10 gcccggctgt gctggctcca gaggatgggc tggccatgtc cctgcatttc atgacattgg 60 gtggcagctc cctgtccccc accgagggca aaggctctgg gctccaaggc cacatcatcg 120 agaacccaca atacttcagt gatgcctgtg ttcaccacat caagcgccgg gacatcgtgc 180 tcaagtggga gctgggggag ggcgcctttg ggaaggtctt ccttgctgag tgccacaacc 240 tcctgcctga gcaggacaag atgctggtgg ctgtcaaggc actgaaggag gcgtccgaga 300 gtgctcggca ggacttccag cgtgaggctg agctgctcac catgctgcag caccagcaca 360 tcgtgcgctt cttcggcgtc tgcaccgagg gccgccccct gctcatggtc tttgagtata 420 tgcggcacgg ggacctcaac cgcttcctcc gatcccatgg acctgatgcc aagctgctgg 480 ctggtgggga ggatgtggct ccaggccccc tgggtctggg gcagctgctg gccgtggcta 540 gccaggtcgc tgcggggatg gtgtacctgg cgggtctgca ttttgtgcac cgggacctgg 600 ccacacgcaa ctgtctagtg ggccagggac tggtggtcaa gattggtgat tttggcatga 660 gcagggatat ctacagcacc gactattacc gtgtgggagg ccgcaccatg ctgcccattc 720 gctggatgcc gcccgagagc atcctgtacc gtaagttcac caccgagagc gacgtgtgga 780 gcttcggcgt ggtgctctgg gagatcttca cctacggcaa gcagccctgg taccagctct 840 ccaacacgga ggcaatcgac tgcatcacgc agggacgtga gttggagcgg ccacgtgcct 900 gcccaccaga ggtctacgcc atcatgcggg gctgctggca gcgggagccc cagcaacgcc 960 acagcatcaa ggatgtgcac gcccggctgc aagccctggc ccaggcacct cctgtctacc 1020 tggatgtcct gggctag 1037 <210> 11 <211> 17 <212> DNA <213> NTRK1 3 'terminal cleavage site seq <400> 11 gcccggctgt gctggct 17 <210> 12 <211> 890 <212> DNA <213> NTRK1 fragment cDNA <400> 12 gtgttcacca catcaagcgc cgggacatcg tgctcaagtg ggagctgggg gagggcgcct 60 ttgggaaggt cttccttgct gagtgccaca acctcctgcc tgagcaggac aagatgctgg 120 tggctgtcaa ggcactgaag gaggcgtccg agagtgctcg gcaggacttc cagcgtgagg 180 ctgagctgct caccatgctg cagcaccagc acatcgtgcg cttcttcggc gtctgcaccg 240 agggccgccc cctgctcatg gtctttgagt atatgcggca cggggacctc aaccgcttcc 300 tccgatccca tggacctgat gccaagctgc tggctggtgg ggaggatgtg gctccaggcc 360 ccctgggtct ggggcagctg ctggccgtgg ctagccaggt cgctgcgggg atggtgtacc 420 tggcgggtct gcattttgtg caccgggacc tggccacacg caactgtcta gtgggccagg 480 gactggtggt caagattggt gattttggca tgagcaggga tatctacagc accgactatt 540 accgtgtggg aggccgcacc atgctgccca ttcgctggat gccgcccgag agcatcctgt 600 accgtaagtt caccaccgag agcgacgtgt ggagcttcgg cgtggtgctc tgggagatct 660 tcacctacgg caagcagccc tggtaccagc tctccaacac ggaggcaatc gactgcatca 720 cgcagggacg tgagttggag cggccacgtg cctgcccacc agaggtctac gccatcatgc 780 ggggctgctg gcagcgggag ccccagcaac gccacagcat caaggatgtg cacgcccggc 840 tgcaagccct ggcccaggca cctcctgtct acctggatgt cctgggctag 890 <210> 13 <211> 17 <212> DNA <213> NTRK1 3 'terminal cleavage site seq <400> 13 gtgttcacca catcaag 17 <210> 14 <211> 796 <212> PRT <213> NTRK1 full length <400> 14 Met Leu Arg Gly Gly Arg Arg Gly Gln Leu Gly Trp His Ser Trp Ala   1 5 10 15 Ala Gly Pro Gly Ser Leu Ala Trp Leu Ile Leu Ala Ser Ala Gly              20 25 30 Ala Ala Pro Cys Pro Asp Ala Cys Cys Pro His Gly Ser Ser Gly Leu          35 40 45 Arg Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His His Leu Pro Gly      50 55 60 Ala Glu Asn Leu Thr Glu Leu Tyr Ile Glu Asn Gln Gln His Leu Gln  65 70 75 80 His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn Leu                  85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe His             100 105 110 Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu         115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Glu Leu Val     130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Arg Trp Leu Gln 145 150 155 160 Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Pro Glu Gln Lys Leu Gln                 165 170 175 Cys His Gly Gln Gly Pro Leu Ala His Met Pro Asn Ala Ser Cys Gly             180 185 190 Val Pro Thr Leu Lys Val Gln Val Pro Asn Ala Ser Val Asp Val Gly         195 200 205 Asp Val Leu Leu Arg Cys Gln Val Glu Gly Arg Gly Leu Glu Gln     210 215 220 Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala Thr Val Met Lys 225 230 235 240 Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser                 245 250 255 Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly             260 265 270 Arg Ala Glu Val Ser Val Glu Val Asn Val Ser Phe Pro Ala Ser Val         275 280 285 Gln Leu His Thr Ala Val Glu Met His His Trp Cys Ile Pro Phe Ser     290 295 300 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser 305 310 315 320 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Ala                 325 330 335 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln Pro Thr             340 345 350 His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly         355 360 365 Gln Ala Ser Ala Ser Ale Ala Phe Met Asp Asn Pro Phe Glu     370 375 380 Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val Asp Thr 385 390 395 400 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe                 405 410 415 Gly Val Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu             420 425 430 Ser Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe         435 440 445 Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met     450 455 460 Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu 465 470 475 480 Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr                 485 490 495 Phe Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu             500 505 510 Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu         515 520 525 Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys     530 535 540 Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu 545 550 555 560 Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe                 565 570 575 Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met             580 585 590 Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala         595 600 605 Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu     610 615 620 Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Gly Met Val Tyr 625 630 635 640 Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys                 645 650 655 Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser             660 665 670 Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met         675 680 685 Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe     690 695 700 Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile 705 710 715 720 Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala                 725 730 735 Ile Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys             740 745 750 Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro         755 760 765 Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu     770 775 780 Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 785 790 795 <210> 15 <211> 397 <212> PRT <213> NTRK1 fragment <400> 15 Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro   1 5 10 15 Phe Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe              20 25 30 Leu Ser Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys          35 40 45 Phe Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala      50 55 60 Met Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr  65 70 75 80 Glu Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln                  85 90 95 Tyr Phe Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val             100 105 110 Leu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala         115 120 125 Glu Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val     130 135 140 Lys Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg 145 150 155 160 Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe                 165 170 175 Phe Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr             180 185 190 Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp         195 200 205 Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly     210 215 220 Leu Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val 225 230 235 240 Tyr Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn                 245 250 255 Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met             260 265 270 Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr         275 280 285 Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys     290 295 300 Phe Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu 305 310 315 320 Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu                 325 330 335 Ala Ile Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala             340 345 350 Cys Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu         355 360 365 Pro Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala     370 375 380 Leu Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 385 390 395 <210> 16 <211> 344 <212> PRT <213> NTRK1 fragment <400> 16 Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser Leu His Phe   1 5 10 15 Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly Lys Gly Ser              20 25 30 Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala          35 40 45 Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu Leu      50 55 60 Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu  65 70 75 80 Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu                  85 90 95 Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu             100 105 110 Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe Gly Val Cys Thr         115 120 125 Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp     130 135 140 Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala 145 150 155 160 Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu                 165 170 175 Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr Leu Ala Gly Leu             180 185 190 His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gln         195 200 205 Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser Ser Asp Ile Tyr     210 215 220 Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu Pro Ile Arg 225 230 235 240 Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe Thr Thr Glu Ser                 245 250 255 Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly             260 265 270 Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala Ile Asp Cys Ile         275 280 285 Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val     290 295 300 Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg His 305 310 315 320 Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro                 325 330 335 Pro Val Tyr Leu Asp Val Leu Gly             340 <210> 17 <211> 5 <212> PRT <213> NTRK1 C terminus cleavage site aa seq <400> 17 Pro Ala Val Leu Ala   1 5 <210> 18 <211> 295 <212> PRT <213> NTRK1 fragment site <400> 18 Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu Leu Gly   1 5 10 15 Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu Leu              20 25 30 Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu Ala          35 40 45 Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu Thr      50 55 60 Met Leu Gln His Gln His Ile Val Arg Phe Phe Gly Val Cys Thr Glu  65 70 75 80 Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp Leu                  85 90 95 Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala Gly             100 105 110 Gly Asp Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu Ala         115 120 125 Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr Leu Ala Gly Leu His     130 135 140 Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gln Gly 145 150 155 160 Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser Ser Asp Ile Tyr Ser                 165 170 175 Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu Pro Ile Arg Trp             180 185 190 Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe Thr Thr Glu Ser Asp         195 200 205 Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys     210 215 220 Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala Ile Asp Cys Ile Thr 225 230 235 240 Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val Tyr                 245 250 255 Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg His Ser             260 265 270 Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro Pro         275 280 285 Val Tyr Leu Asp Val Leu Gly     290 295 <210> 19 <211> 5 <212> PRT <213> NTRK1 C terminus cleavage site seq <400> 19 Val His His Ile Lys   1 5 <210> 20 <211> 5 <212> PRT <213> NTRK1 C terminus cleavage site seq <400> 20 Val His His Ile Lys   1 5 <210> 21 <211> 33 <212> DNA <213> TPM3-NTRK1 fusion site seq <400> 21 attgatgacc tggaagacac taacagcaca tct 33 <210> 22 <211> 33 <212> DNA <213> TPM3-NTRK1 fusion site seq <400> 22 attgatgacc tggaaggccc ggctgtgctg gct 33 <210> 23 <211> 33 <212> DNA <213> TPM3-NTRK1 fusion site seq <400> 23 attgatgacc tggaaggtgt tcaccacatc aag 33 <210> 24 <211> 11 <212> PRT <213> TPM3-NTRK1 fusion site aa seq <400> 24 Ile Asp Asp Leu Glu Asp Thr Asn Ser Thr Ser   1 5 10 <210> 25 <211> 11 <212> PRT <213> TPM3-NTRK1 fusion site aa seq <400> 25 Ile Asp Asp Leu Glu Gly Pro Ala Val Leu Ala   1 5 10 <210> 26 <211> 11 <212> PRT <213> TPM3-NTRK1 fusion site aa seq <400> 26 Ile Asp Asp Leu Glu Gly Val His His Ile Lys   1 5 10 <210> 27 <211> 1995 <212> DNA <213> LMNA CDS <400> 27 atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60 ctgtcgccca cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat 120 cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc 180 cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240 tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc 300 cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360 accaagaagg agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg 420 ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc 480 gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540 aagcaacttc aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg 600 aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga gaccaagcgc 660 cgtcatgaga cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720 ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag 780 aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840 aacagcaacc tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac 900 agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga ggcgaagctt 960 cgagacctgg aggactcact ggcccgtgag cgggacacca gccggcggct gctggcggaa 1020 aaggagcggg agatggccga gatgcgggca aggatgcagc agcagctgga cgagtaccag 1080 gagcttctgg acatcaagct ggccctggac atggagatcc acgcctaccg caagctcttg 1140 gagggcgagg aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc 1200 cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa aaagcgcaaa 1260 ctggagtcca ctgagagccg cagcagcttc tcacagcacg cacgcactag cgggcgcgtg 1320 gccgtggagg aggtggatga ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag 1380 gaccagtcca tgggcaattg gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440 taccggttcc caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca 1500 ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca gaacacctgg 1560 ggctgcggga acagcctgcg tacggctctc atcaactcca ctggggaaga agtggccatg 1620 cgcaagctgg tgcgctcagt gactgtggtt gaggacgacg aggatgagga tggagatgac 1680 ctgctccatc accaccacgg ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740 ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc 1800 agcggctcag gagcccaggt gggcggaccc atctcctctg gctcttctgc ctccagtgtc 1860 acggtcactc gcagctaccg cagtgtgggg ggcagtgggg gtggcagctt cggggacaat 1920 ctggtcaccc gctcctacct cctgggcaac tccagccccc gaacccagag cccccagaac 1980 tgcagcatca tgtaa 1995 <210> 28 <211> 985 <212> DNA <213> LMNA fragment cDNA <400> 28 atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60 ctgtcgccca cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat 120 cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc 180 cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240 tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc 300 cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360 accaagaagg agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg 420 ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc 480 gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540 aagcaacttc aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg 600 aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga gaccaagcgc 660 cgtcatgaga cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720 ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag 780 aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840 aacagcaacc tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac 900 agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga ggcgaagctt 960 cgagacctgg aggactcact ggccc 985 <210> 29 <211> 16 <212> DNA <213> LMNA 5 'terminal cleavage site seq <400> 29 gaggactcac tggccc 16 <210> 30 <211> 664 <212> PRT <213> LMNA full length <400> 30 Met Glu Thr Pro Ser Gln Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala   1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr Arg Ile Thr Arg Leu Gln Glu Lys              20 25 30 Glu Asp Leu Gln Glu Leu Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg          35 40 45 Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr      50 55 60 Glu Ser Glu Glu Val Ser Ser Glu Val Ser Gly Ile Lys Ala Ala  65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala                  85 90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu Glu             100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly Asp Leu Ile         115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys     130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu                 165 170 175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp             180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys         195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr     210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val                 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp             260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala         275 280 285 His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala     290 295 300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg                 325 330 335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met             340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys Leu Ala         355 360 365 Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu Glu Gly Glu Glu     370 375 380 Glu Arg Leu Arg Leu Ser Pro Ser Ser Thr Ser Gln Arg Ser Ser Arg Gly 385 390 395 400 Arg Ala Ser Ser Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr                 405 410 415 Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln             420 425 430 His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu         435 440 445 Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met     450 455 460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr 465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln Val Val Thr                 485 490 495 Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser Pro Thr Asp Leu             500 505 510 Val Trp Lys Ala Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr         515 520 525 Ala Leu Ile Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val     530 535 540 Arg Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550 555 560 Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro                 565 570 575 Ala Glu Tyr Asn Leu Arg Ser Ser Thr Val Leu Cys Gly Thr Cys Gly             580 585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Val Gly         595 600 605 Gly Pro Ile Ser Ser Gly Ser Ser Ala Ser Ser Val Thr Val Thr Arg     610 615 620 Ser Tyr Arg Ser Val Gly Gly Ser Gly Gly Gly Ser Phe Gly Asp Asn 625 630 635 640 Leu Val Thr Arg Ser Tyr Leu Leu Gly Asn Ser Ser Pro Arg Thr Gln                 645 650 655 Ser Pro Gln Asn Cys Ser Ile Met             660 <210> 31 <211> 328 <212> PRT <213> LMNA fragment <400> 31 Met Glu Thr Pro Ser Gln Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala   1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr Arg Ile Thr Arg Leu Gln Glu Lys              20 25 30 Glu Asp Leu Gln Glu Leu Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg          35 40 45 Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr      50 55 60 Glu Ser Glu Glu Val Ser Ser Glu Val Ser Gly Ile Lys Ala Ala  65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala                  85 90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu Glu             100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly Asp Leu Ile         115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys     130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu                 165 170 175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp             180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys         195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr     210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val                 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp             260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala         275 280 285 His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala     290 295 300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala                 325 <210> 32 <211> 5 <212> PRT <213> LMNA N terminus cleavage site seq <400> 32 Glu Asp Ser Leu Ala   1 5 <210> 33 <211> 33 <212> DNA <213> LMNA-NTRK1 fusion site seq <400> 33 gaggactcac tggcccgccc ggctgtgctg gct 33 <210> 34 <211> 11 <212> PRT <213> LMNA-NTRK1 fusion site aa seq <400> 34 Glu Asp Ser Leu Ala Gly Pro Ala Val Leu Ala   1 5 10 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TPM3-NTRK1 Forward Primer Sequence <400> 35 aagaagataa atatgagg 18 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TPM3-NTRK1 Reverse Primer Sequence <400> 36 ccgtgccgca tatactcaaa 20 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LMNA-NTRK1 Forward Primer Sequence <400> 37 ccaggtggag cagtataaga ag 22 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMNA-NTRK1 Reverse Primer Sequence <400> 38 tgtgggttct cgatgatgtg 20

Claims (42)

A fusion protein comprising the following structural formula:
A fragment containing the N-terminus of N- [LMNA (Lamin A)] - [a fragment containing the C-terminus of NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1]
A fragment containing the N-terminus of N- [TPM3 (Tropomyosin 3)] - [a fragment containing the C-terminus of NTRK1] -C.
The fusion protein according to claim 1, wherein the LMNA, TPM3 or NTRK1 is derived from human.
3. The fusion protein according to claim 2, wherein the human-derived LMNA comprises the amino acid sequence of SEQ ID NO: 27.
2. The fusion protein according to claim 1, wherein the fragment comprising the N-terminus of the LMNA comprises a first to 328th amino acid from the N-terminus in the human-derived LMNA protein.
2. The fusion protein of claim 1, wherein the fragment comprising the N-terminus of the LMNA comprises the amino acid sequence of SEQ ID NO: 31.
3. The fusion protein according to claim 2, wherein the human-derived TPM3 comprises the amino acid sequence of SEQ ID NO: 4.
The fusion protein according to claim 1, wherein the fragment comprising the N-terminus of TPM3 comprises amino acids 1 to 258 from the N-terminus in the human-derived TPM3 protein.
The fusion protein of claim 1, wherein the fragment comprising the N-terminus of TPM3 is comprised of the amino acid sequence of SEQ ID NO: 5.
3. The fusion protein of claim 2, wherein the human-derived NTRK1 comprises the amino acid sequence of SEQ ID NO: 7.
The fragment of claim 1, wherein the fragment comprising the C-terminus of NTRK1 comprises the amino acid sequence from the N-terminus to the N-terminus of the human NTRK1 protein, from amino acid 453 to amino acid 796 or amino acid from position 502 to position 796 Phosphorylated fusion protein.
The fusion protein of claim 1, wherein the fragment comprising the C-terminus of NTRK1 is comprised of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19.
A polynucleotide encoding the fusion protein of claim 1.
A fusion gene polynucleotide consisting of the following structural formula:
5 '- [fragment containing the 5' end of the LMNA gene] - [fragment containing the 3 'end of the NTRK1 gene] -3'
5 '- [Fragment containing the 5' end of TPM3] - [Fragment containing the 3 'end of the NTRK1 gene] -3'.
14. The TRK1 fusion gene polynucleotide according to claim 13, wherein the LMNA gene is a nucleic acid sequence of SEQ ID NO: 28.
14. The TRK1 fusion gene polynucleotide according to claim 13, wherein the TPM3 gene is composed of the nucleotide sequence of SEQ ID NO: 2.
14. The TRK1 fusion gene polynucleotide according to claim 13, wherein the NTRK1 gene is composed of the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 13.
13. A colon cancer diagnostic composition comprising the NTRK1 fusion protein according to claim 1, the fusion gene according to claim 13, or an agent for measuring the transcription product of said fusion gene.
18. The composition for diagnosing colorectal cancer according to claim 17, wherein the transcription product of the fusion gene is mRNA.
19. The composition for diagnosing colorectal cancer according to claim 18, wherein the agent for measuring the mRNA of the fusion gene comprises a primer that specifically binds to the mRNA of the fusion gene.
[Claim 21] The method according to claim 19, wherein the primer specifically binding to the mRNA of the fusion gene is a primer designed so that a region amplified by the primer includes a fusion point of two genes to be fused, A composition for cancer diagnosis.
21. The method according to claim 20, wherein when the two genes to be fused are the TPM3 gene and the NTRK1 gene, a primer specific to the nucleotide sequence encoding the N-terminal of the TPM3 protein and a primer specific to the nucleotide sequence encoding the C- Wherein the composition is a primer pair.
The composition for diagnosing colorectal cancer according to claim 21, wherein the primer specifically binding to the mRNA of the fusion gene is a primer pair consisting of the nucleotide sequence of SEQ ID NO: 35 and SEQ ID NO: 36.
The method according to claim 19, wherein when the two genes to be fused are the LMNA gene and the NTRK1 gene, a primer specific to the nucleotide sequence encoding the N-terminal of the LMNA protein and a primer specific to the nucleotide sequence encoding the C- Wherein the composition is a primer pair.
24. The composition for diagnosing colorectal cancer according to claim 23, wherein the primer that specifically binds to the mRNA of the fusion gene is a primer pair consisting of the nucleotide sequence of SEQ ID NO: 37 and SEQ ID NO: 38.
18. The composition for diagnosing colorectal cancer according to claim 17, wherein the agent for measuring the fusion gene comprises a probe or a primer that specifically binds to the fusion gene.
18. The composition for diagnosing colorectal cancer according to claim 17, wherein the agent for measuring the NTRK1 fusion protein comprises an antibody specific to the protein.
27. The composition for diagnosing colorectal cancer according to claim 26, wherein the antibody specific to the NTRK1 fusion protein binds to the C terminus of the NTRK1 protein.
17. A colon cancer diagnostic kit comprising the composition of claim 17.
29. The diagnostic kit for colon cancer according to claim 28, which is an RT-PCR kit, DNA chip kit or protein chip kit.
Measuring the level of the NTRK1 fusion gene, its transcription product or its protein from a separate sample of individuals suspected of having colon cancer; And comparing the measured expression level of the fusion gene, its transcription product or its protein with a fusion gene of a normal control sample, a transcription product thereof or a protein expression level thereof.
31. The method of claim 30, wherein the NTRK1 fusion gene comprises a polynucleotide that constitutes the 5 ' end of the LMNA gene or the TPM3 gene; And a polynucleotide that constitutes the 3 ' end of the NTRK1 gene.
31. The method of claim 30, wherein the level of the fusion gene, its transcription product, or protein expression thereof measured from a separate sample of a subject suspected of having colorectal cancer is higher than the level of the fusion gene, transcription product thereof or protein expression thereof of a normal control sample, A method for providing information necessary for diagnosis of colorectal cancer, which comprises diagnosing colon cancer.
Measuring the level of the NTRK1 fusion gene, its transcription product or its protein from a separate sample of colon cancer patients; And comparing the measured expression level of the fusion gene, its transcription product, or its protein with a fusion gene of a normal control sample, a transcription product thereof or a protein expression level thereof, to provide information necessary for predicting colorectal cancer treatment prognosis How to.
34. The method of claim 33, wherein the level of expression of the fusion gene, its transcription product, or protein thereof from the isolated sample of a colon cancer patient is higher than the fusion gene of the normal control sample, the transcription product thereof or the protein expression thereof, Wherein the predicted outcome is predicted to be worse than that when the same or lower is used.
34. The method of claim 33, wherein the level of expression of the fusion gene, its transcription product, or protein thereof from a separate sample of a patient with colorectal cancer is higher than the level of the fusion gene, transcription product thereof or protein expression thereof of a normal control sample, A method for providing information necessary for predicting colorectal cancer treatment prognosis, which is predicted to be highly effective in colorectal cancer treatment.
Treating a candidate cancer cell cancer candidate cell with a cell expressing the NTRK1 fusion gene; And measuring the expression level of the NTRK1 fusion gene.
37. The screening method for a colorectal cancer therapeutic agent according to claim 36, wherein when the expression level of the NTRK1 fusion gene is lowered by treatment with a candidate substance for treatment of colorectal cancer, it is judged to be a therapeutic agent for colon cancer.
A pharmaceutical composition for the prophylaxis or treatment of colorectal cancer comprising an NTRK1 fusion protein inhibitor as an active ingredient.
The pharmaceutical composition according to claim 38, wherein the NTRK1 fusion protein inhibitor is ARRY-470, a compound represented by the following general formula (1) or a compound represented by the general formula (2).
[Chemical Formula 1]

(2)

39. The pharmaceutical composition according to claim 38, wherein the NTRK1 fusion protein inhibitor is an NTRK1 fusion gene expression inhibitor.
41. The composition of claim 40, wherein the NTRK1 fusion gene expression inhibitor is selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the NTRK1 fusion gene.
39. The composition of claim 38, wherein the NTRK1 fusion protein inhibitor is an antibody that specifically binds to the NTRK1 fusion protein.

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