Biomarker composition comprising AMPD3 for predicting sensitivity to therapeutic agent for neurofibromatosis, and pharmaceutical composition comprising AMPD3 inhibitor for preventing or treating neurofibromatosis

Abstract

The present invention relates to: a biomarker composition comprising AMPD3 for predicting sensitivity to a therapeutic agent for neurofibromatosis (NF1); a pharmaceutical composition comprising an AMPD3 inhibitor for preventing or treating NF1; and use thereof. The composition according to one aspect is highly useful as a diagnostic marker for therapeutic agent sensitivity in patients with NF1, and can be used as a new therapeutic target for NF1.

Description

Biomarker composition for predicting sensitivity to neurofibromatosis treatment agent including AMPD3 and pharmaceutical composition for preventing or treating neurofibromatosis including AMPD3 inhibitor

[0001] The present invention relates to a biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent including AMPD3, a pharmaceutical composition for preventing or treating neurofibromatosis including an AMPD3 inhibitor, and a use thereof.

[0002] Neurofibromatosis Type 1 (NF1), first described by Fredrich von Recklin-ghausen in 1882, is an autosomal dominant inherited disorder characterized by tumors forming along the peripheral nerves, occurring in approximately 1 in 3,500 people. These tumors can develop anywhere in the body where nerves are present, and because they are often multiple, they are called "neurofibromatosis."

[0003] The main symptoms include neurofibromas, café-au-lait spots, Lisch nodules, optic pathway glioma, scoliosis, tibial dysplasia, plexiform neurofibromas (PN), and pontine glioma, which can occur anywhere there is a nerve.

[0004] Neurofibromatosis type 1 is caused by mutations in the NF1 gene, and approximately 50% of patients develop the disease due to spontaneous, rather than inherited, mutations. The NF1 gene is located on chromosome 17q11.2, and consists of 350 kb of genomic DNA, 57 exons, and mRNA of approximately 11 to 13 kb. The tumor suppressor gene NF1 encodes a 320 kDa cytoplasmic protein, neurofibromin, which contains a GTPase-activating protein (GAP)-related domain (GRD) region at amino acids 1125 to 1537 that downregulates Ras signaling. GAP regulates Ras activity by stimulating the intrinsic GTPase of Ras to convert it from the active form, Ras-GTP, to the inactive form, Ras-GDP. The Ras protein, encoded by Ras, is expressed in both neural crest cells and endothelial cells, and its signals are transduced through various downstream signaling pathways. Therefore, loss or dysfunction of neurofibromin leads to the development of neurofibromatosis type 1 (NF1) by hyperactivation and accumulation of RAS, leading to severe, life-threatening complications in approximately 20% of patients.

[0005] Approximately 8-13% of patients with neurofibromatosis develop malignant transformation, resulting in unpredictable and severe clinical symptoms. However, due to its hereditary nature, it is incurable. When malignant neurofibrosarcoma develops, conventional treatment, including tumor resection, is the only treatment. However, depending on the tumor's location, surgery is often impossible. Therefore, regardless of the stage of cancer progression, the development of neurofibrosarcoma is directly linked to the patient's life.

[0006] The clinical phenotypes of neurofibromatosis type 1 (NF1) and the precise mechanisms underlying its malignant transformation remain unclear. Representative treatments include radiotherapy, surgical resection, and cytotoxic agents, but long-term efficacy is limited. Therefore, new therapeutic targets and candidate agents utilizing novel approaches are needed.

[0007] Accordingly, we discovered a diagnostic marker that can predict the therapeutic sensitivity of neurofibromatosis type 1 (NF1) and a new therapeutic target for neurofibromatosis, and confirmed the possibility of using the therapeutic target inhibitor as a new neurofibromatosis treatment.

[0008] One aspect is to provide a biomarker composition for predicting sensitivity to a neurofibromatosis type 1 (NF1) treatment agent, comprising a preparation capable of measuring the expression level of AMPD3 (Adenosine monophosphate deaminase) protein or a gene encoding the same.

[0009] Another aspect is to provide a biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent further comprising an inhibitor of expression or activity of TGFBR3 (TGF-beta receptor 3).

[0010] Another aspect is to provide a kit for predicting sensitivity to a neurofibromatosis treatment agent comprising the above biomarker composition.

[0011] Another aspect provides a method for providing information necessary for predicting sensitivity to a neurofibromatosis treatment agent, comprising the steps of: measuring the expression level of AMPD3 protein or a gene encoding it in a biological sample obtained from an individual; and / or measuring the expression level of TGFBR3 protein or a gene encoding it.

[0012] Another aspect is to provide a pharmaceutical composition for preventing or treating neurofibromatosis comprising an inhibitor of expression or activity of AMPD3 (Adenosine monophosphate deaminase) and / or an inhibitor of expression or activity of TGFBR3 (TGF-beta receptor 3).

[0013] A kit for preventing or treating neurofibromatosis is provided, comprising an inhibitor of AMPD3 expression or activity and / or an inhibitor of TGFBR3 (TGF-beta receptor 3) expression or activity.

[0014] Another aspect provides the use of an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase) for preventing or treating neurofibromatosis.

[0015] Another aspect provides a method for preventing or treating neurofibromatosis, comprising administering to a subject in need thereof an effective amount of an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase).

[0016] Another aspect provides the use of an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase) for use in the manufacture of a pharmaceutical preparation for the prevention or treatment of neurofibromatosis.

[0017] To achieve the above purpose, one aspect provides a biomarker composition for predicting sensitivity to a neurofibromatosis type 1 (NF1) treatment agent, comprising a preparation capable of measuring the expression level of AMPD3 (Adenosine monophosphate deaminase) protein or a gene encoding the same.

[0018] The term "AMPD3 (Adenosine monophosphate deaminase)" used herein refers to an enzyme that deaminates AMP into inosine monophosphate (IMP), and is involved in the regulation of energy metabolism of the purine system. AMPD3 is known to regulate GTP synthesis, which can increase the activity of GTP-bound RAS as a source of active Ras. Specifically, AMPD3 promotes the synthesis of GTP, which is a resource of the active form of RAS (GTP-bound RAS), and thus AMPD3 can directly or indirectly affect the activity of neurofibromin. For example, overexpressed AMPD inhibits the anticancer activity of neurofibromin, which regulates the MEK-ERK signaling pathway in association with overactive RAS, whereas AMPD3 inhibition restores neurofibromin expression and exhibits anticancer activity. Additionally, the above AMPD3 is known to be involved in tumor malignancy due to its positive correlation with receptor tyrosine kinase (KIT) in gastrointestinal stromal tumors (GIST).

[0019] The term "susceptibility" in this specification refers to whether a particular drug is therapeutically effective for an individual patient with neurofibromatosis. Even for treatments recognized as effective for neurofibromatosis, it is known that some patients respond to the drug while others do not. Whether a treatment is effective for an individual patient with neurofibromatosis can be referred to as "susceptibility to the drug."

[0020] The term "diagnosis" as used herein means confirming the presence or characteristics of a pathological condition, including determining an individual's susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining a prognosis for an individual with a particular disease.

[0021] The term "biomarker" in this specification may also be used as a companion diagnostic marker, and refers to a substance that can predict in advance the responsiveness of a patient to a specific drug treatment in a biological sample. It may include organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (e.g., monosaccharides, disaccharides, oligosaccharides, etc.), which show an increase or decrease in a biological sample collected from a neurofibromatosis patient compared to a biological sample obtained from a normal individual.

[0022] In one specific example, the biomarker for predicting sensitivity to the neurofibromatosis treatment agent may be the adenosine monophosphate deaminase (AMPD3) protein or a gene encoding it. If its expression level is higher than that of a normal control group, sensitivity to the neurofibromatosis treatment agent may be determined to be low. Conversely, if its expression level is low, sensitivity to the neurofibromatosis treatment agent may be determined to be high.

[0023] The above neurofibromatosis treatment agent may be a biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent, which is a compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof:

[0024] [Chemical Formula 1]

[0025]

[0026] In the above formula

[0027] Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl;

[0028] R is hydrogen or halogen.

[0029] The term "halogen" means F, Cl, Br or I unless otherwise stated.

[0030] The term "hydroxyl" means a monovalent group consisting of one hydrogen atom and one oxygen atom, with the chemical formula -OH, unless otherwise stated.

[0031] The term "alkyl", unless otherwise specified, refers to a linear or branched saturated hydrocarbon residue. For example, "C1-10 alkyl" refers to an alkyl having a skeleton of 1 to 10 carbons. Specifically, C1-10 alkyl can include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.

[0032] The term "alkoxy", unless otherwise specified, means a group having the formula -O-alkyl, wherein the alkyl group as defined above is attached to the parent compound via an oxygen atom. The alkyl portion of the alkoxy group may have 1 to 20 carbon atoms (i.e., C1-C20 alkoxy), 1 to 12 carbon atoms (i.e., C1-C12 alkoxy), or 1 to 6 carbon atoms (i.e., C1-C6 alkoxy). Examples of suitable alkoxy groups include methoxy (-O-CH3 or -OMe), ethoxy (-OCH2CH3 or -OEt), t-butoxy (-OC(CH3)3 or -O-tBu), and the like.

[0033] The term "aryl" refers to an aromatic hydrocarbon radical derived by the removal of one hydrogen atom from six carbon atoms of a parent aromatic ring system. For example, an aryl group may have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms.

[0034] The term "cycloalkyl" refers to a saturated monocycle or polycycle containing only carbon atoms in the ring. A cycloalkyl may have 3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycloalkyl, or up to about 20 carbon atoms as a polycycle.

[0035] The term "heterocycle" refers to an aromatic or non-aromatic ring having one or more heteroatoms, which may be saturated or unsaturated, and may be monocyclic or polycyclic. For example, a "4- to 10-membered heterocycle" refers to a heterocycle whose skeleton is composed of a total of 4 to 10 atoms, including heteroatoms and carbon atoms. Specifically, a 4- to 10-membered heterocycle may include azetidine, diazetidine, pyrrolidine, pyrrole, imidazolidine, imidazole, pyrazolidine, pyrazole, oxazolidine, oxazole, isoxazolidine, isoxazole, thiazolidine, thiazole, isothiazolidine, isothiazole, piperidine, pyridine, piperazine, diazine, morpholine, thiomorpholine, azepane, diazepane, and the like.

[0036] The term "heteroaryl" refers to an aromatic heterocyclyl having one or more heteroatoms in the ring. Non-limiting examples of heteroaryls include pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, and pyrazyl (which may have one or more substituents in the ring).

[0037] The term "heterocycloalkyl" refers to a non-aromatic heterocyclyl having one or more heteroatoms in the ring. Heterocycloalkyls may have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring, as long as the presence of the double bonds renders the ring non-aromatic. Non-limiting examples of heterocycloalkyls include azetidinyl, aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, homopiperazinyl, morpholino, thiomorpholino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl (which may have one or more substituents in the ring).

[0038] The term "heteroatom" means an atom other than carbon (C), specifically a nitrogen (N), oxygen (O), or sulfur (S) atom.

[0039] The term "substitution" refers to the replacement of a hydrogen atom within a molecular structure with a substituent, such that the resulting compound is chemically stable without exceeding the valence of the designated atom. For example, "group A is replaced with substituent B" may mean that a hydrogen atom bonded to an atom such as carbon that constitutes the skeleton of group A is replaced with substituent B, so that group A and substituent B form a covalent bond.

[0040] The above Z is p-tolyl; (4-chloromethyl-phenyl); (2-chloro-pyridin-3-yl); (2-fluoro-phenyl); (3,4-difluoro-phenyl); (4-methoxy-phenyl); benzo[1,3]dioxol-yl; (4-ethyl-phenyl); o-tolyl; (2-chloro-phenyl); (3-methyl-thiophen-2-yl); benzo[b]thiophen-2-yl; (3-fluoro-phenyl); (4-tert-butyl-phenyl); (2-methoxy-phenyl); (2,difluoro-phenyl); thiophen-2-yl; (2,4-difluoro-phenyl); (3-chloro-phenyl); m-tolyl; (4-trifluoromethyl-phenyl); (4-fluoro-phenyl); (3-methoxy-phenyl); phenyl; (2,6-difluoro-phenyl); (2,dimethyl-furan-3-yl); (4-pyrrol-1-yl-phenyl); (3-dimethylamino-phenyl); biphenyl-4-yl; (4-dimethylamino-phenyl); benzo[1,2,5]oxadiazol-yl; m-tolyl; (2-trifluoromethyl-phenyl); (6-chloro-pyridin-3-yl); (3,bis-trifluoromethyl-phenyl); furan-2-yl; (4-nitro-phenyl); (3,4-dimethoxy-phenyl); (3-trifluoromethoxy-phenyl); naphthalen-1-yl; cyclohexyl; pyridin-3-yl; pyridin-4-yl; cyclopentyl; cyclopropyl; (4-pentyloxy-phenyl); (3,4,trimethoxy-phenyl); (4-isobutyl-phenyl); cyclobutyl; (1-acetyl-piperidin-4-yl); isoxazol-yl; [2-chloro-6-fluoro-phenyl)-methyl-isoxazol-4-yl] or [2-chloro-phenyl)-methyl-isoxazol-4-yl]. Specifically, Z may be (2-fluoro-phenyl); (3,4-difluoro-phenyl); (2-chloro-phenyl); (3-fluoro-phenyl); (2,5-difluoro-phenyl); (2,4-difluoro-phenyl); (3-chloro-phenyl); (4-fluoro-phenyl); or (2,6-difluoro-phenyl).

[0041] The compound represented by the above chemical formula 1 can read-through premature stop codons.

[0042] The compound represented by the above chemical formula 1 may be 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid. The compound may be represented by the following chemical formula 2:

[0043] [Chemical Formula 2]

[0044]

[0045] The compound represented by the above chemical formula 2 is also called Ataluren, PCT124 or Translarna and is known to read-through premature stop codons.

[0046] The above neurofibromatosis treatment agent comprises a pharmaceutically acceptable salt of the above compound. The pharmaceutically acceptable salt must have low toxicity to humans and must not adversely affect the biological activity and physicochemical properties of the parent compound.

[0047] For example, the pharmaceutically acceptable salt may be an alkali metal salt (such as a sodium salt) or an alkaline earth metal salt (such as a potassium salt). The alkali metal salt or alkaline earth metal salt may be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate.

[0048] Additionally, the compounds may have chiral carbon centers and may therefore exist in the form of R or S isomers, racemates, individual enantiomers or mixtures, individual diastereomers or mixtures, and all such stereoisomers and mixtures thereof may fall within the scope of the present invention.

[0049] Additionally, the compound may include hydrates and solvates of the compound. The hydrates and solvates may be prepared using known methods, and are preferably non-toxic and water-soluble. In particular, the hydrates and solvates may preferably be formed by combining 1 to 5 molecules of water and an alcoholic solvent (particularly, ethanol, etc.), respectively.

[0050] The measurement of the expression level of the above AMPD3 protein is a process of confirming the presence and expression level of the protein in a biological sample of an individual in order to predict sensitivity to a neurofibromatosis treatment agent, and confirms the amount of the protein using a molecule that specifically binds to the protein.

[0051] The above "agent capable of measuring the expression level of a protein" may be at least one selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, and a nanoparticle, but is not limited thereto.

[0052] The analytical methods for this purpose include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, immunohistochemical analysis, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, two-dimensional electrophoresis analysis, protein mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, etc. are available, but are not limited thereto.

[0053] Measurement of the expression level of the gene encoding the above protein is a process of confirming the presence and expression level of the gene encoding the above protein in a biological sample of an individual in order to predict sensitivity to a neurofibromatosis treatment agent, and confirms the amount of the gene using a molecule that specifically binds to the gene.

[0054] The above "agent capable of measuring the expression level of a gene encoding a protein" may be at least one selected from the group consisting of a primer pair, a probe, and an antisense nucleotide that specifically bind to the gene, but is not limited thereto.

[0055] Analytical methods for this include, but are not limited to, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction, RNase protection assay (RPA), Northern blotting, and DNA chips.

[0056] The above neurofibromatosis may have a stop codon mutation in the NF1 gene.

[0057] The term "stop codon mutation" in this specification means that a part of the base sequence becomes one of the stop codons UAG, UAA, or UGA due to mutation, thereby stopping the synthesis of a polypeptide.

[0058] The above-mentioned termination codon mutation may be due to a nonsense mutation or a frameshift mutation, which are point mutations in which a DNA base is substituted with another base.

[0059] The above "nonsense mutation" refers to a mutation in which, when a DNA base sequence is converted into a protein, part of the base sequence is converted into a stop codon (one of UAG, UAA, or UGA), so that protein synthesis no longer occurs.

[0060] The above "frameshift mutation" refers to a mutation in which the amino acid sequence of a protein produced through the transcription and translation process of a gene is significantly changed due to a mutation in which a number of nucleotides is inserted or deleted that is not a multiple of 1, 2, or 3 in the DNA base sequence.

[0061] In one embodiment, the stop codon mutation is i) when cytidine (C), which is the 5242nd base in the DNA sequence of the NF1 gene, is substituted with thymine (T), the 1748th amino acid constituting the NF1 protein, arginine (Arg, R), is converted into a stop codon, ii) when cytidine (C), which is the 2560th base in the DNA sequence of the NF1 gene, is substituted with thymine (T), the 854th amino acid constituting the NF1 protein, glutamine (Gln, Q), is converted into a stop codon, iii) when cytidine (C), which is the 6792nd base in the DNA sequence of the NF1 gene, is substituted with adenine (A), the 2264th amino acid constituting the NF1 protein, tyrosine (Tyr, Y), is converted into a stop codon, iv) when cytidine (C), which is the 3565th base in the DNA sequence of the NF1 gene, It is possible that glutamine (Gln, Q), the 1189th amino acid that constitutes the NF1 protein, was converted into a stop codon by being substituted with thymine (T).

[0062] In another embodiment, the biomarker composition provides a biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent that further comprises an inhibitor of expression or activity of TGFBR3 (TGF-beta receptor 3).

[0063] The term "TGFBR3 (TGF-beta receptor 3)" used herein refers to a membrane proteoglycan that is one of the regulatory factors of the TGF-beta signaling system and functions as a co-receptor with members of the TGF-beta receptor superfamily. The TGFBR3 is known to be involved in the progression of colon cancer and triple-negative breast cancer, and NF1+ / - fibroblasts that respond to TGF-beta signals are known to exhibit a phenotype of neurofibroma due to increased cell proliferation and collagen formation by overactivation of the Ras-c-abl signaling pathway.

[0064] The terms "susceptibility", "biomarker", "agent capable of measuring the expression level of a neurofibromatosis treatment agent", "agent capable of measuring the expression level of a gene encoding a protein", "stop codon mutation", etc. are as described above.

[0065] One aspect provides a kit for predicting sensitivity to a neurofibromatosis treatment agent comprising the above biomarker composition.

[0066] The kit can predict sensitivity to the neurofibromatosis treatment using a biological sample isolated from the subject. Specifically, the kit can predict sensitivity to the neurofibromatosis treatment using fibroblasts collected from the subject.

[0067] The above kit may include not only a preparation for measuring the expression level of the protein or gene, but also tools, reagents, etc. commonly used in immunological analysis.

[0068] Examples of the above tools or reagents may include, but are not limited to, a suitable carrier, a label capable of generating a detectable signal, a chromophore, a solubilizer, a detergent, a buffer, a stabilizer, and the like. When the label is an enzyme, the label may include a substrate capable of measuring enzyme activity and a reaction terminator. The carrier may be a soluble carrier or an insoluble carrier. An example of a soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of an insoluble carrier may be a polymer such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, a fluororesin, cross-linked dextran, a polysaccharide, a metal-plated magnetic microparticle on latex, other paper, glass, metal, agarose, and combinations thereof.

[0069] The biomarker composition is as described above.

[0070] Another aspect provides a method for providing information necessary for predicting sensitivity to a neurofibromatosis treatment agent, comprising the step of measuring the expression level of AMPD3 protein or a gene encoding it in a biological sample obtained from an individual.

[0071] Another aspect provides a method for providing information necessary for predicting sensitivity to a neurofibromatosis treatment agent, further comprising the step of measuring the expression level of TGFBR3 protein or a gene encoding it.

[0072] The method may further include a step of determining that the sensitivity to the neurofibromatosis treatment agent is high when the expression level of the AMPD3 and / or TGFBR3 protein or the gene encoding the same is similar to or lower than the expression level measured in a biological sample obtained from a normal individual, and conversely, determining that the sensitivity to the neurofibromatosis treatment agent is low when the expression level thereof is high.

[0073] The term "biological sample" means any sample obtained from an individual from which the expression level of AMPD3 and / or TGFBR3 proteins or the gene encoding them can be measured according to one aspect.

[0074] The biological sample may be, but is not limited to, blood, plasma, serum, urine, saliva, sputum, cerebrospinal fluid, cells, cell cultures, tissue extracts, or tumor tissue. Specifically, the biological sample may be fibroblasts or blood collected from an individual. The biological sample may be prepared by processing using a method commonly used in the art.

[0075] Another aspect provides a pharmaceutical composition for preventing or treating neurofibromatosis, comprising an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase).

[0076] The term "AMPD3 (Adenosine monophosphate deaminase) expression or activity inhibitor" as used herein may directly bind to the AMPD3 gene or protein, bind to a ligand of AMPD3, or inhibit binding of AMPD3 to its ligand. Through this, the expression or activity of AMPD3 can be suppressed or inhibited, and ultimately the growth or death of a tumor can be inhibited. The efficacy of an AMPD3 inhibitor is, for example, 50% (half-maximal inhibitor concentration or IC 50 ) can be measured by the concentration of AMPD3 inhibitor.

[0077] The above ligand refers to a substance that forms a complex by binding to a biomolecule such as a receptor protein to perform a biological purpose, and includes a substrate, activator, signaling lipid, neurotransmitter, etc.

[0078] The term "inhibitor" may be used interchangeably with "suppressant," and "inhibition" may also be used interchangeably with "suppression." The terms "inhibition" or "suppression" refer to a decrease in the activity, binding, or expression of a polypeptide.

[0079] The term "prevention" as used herein refers to any action that suppresses or delays cancer by administering a pharmaceutical composition according to the present invention. Furthermore, the term "treatment" as used herein refers to any action that improves or completely cures cancer by administering a pharmaceutical composition according to the present invention.

[0080] According to one specific example, the AMPD3 expression inhibitor may be at least one selected from the group consisting of antisense nucleic acids, siRNA, shRNA, miRNA, and ribozymes that complementarily bind to DNA or mRNA of AMPD3.

[0081] The term "expression inhibition" as used herein encompasses the inhibition of gene transcription and translation into protein. Furthermore, it encompasses both complete cessation of gene expression and reduced expression.

[0082] As used herein, the term "antisense nucleic acid" means DNA, RNA or a fragment or derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and capable of binding or hybridizing complementarily to the sequence of the mRNA to inhibit translation of the mRNA into protein.

[0083] As used herein, the term "siRNA (small interfering RNA)" refers to a short double-stranded RNA that can induce RNA interference (RNAi) by cleaving a specific mRNA. siRNA comprises a sense RNA strand with a sequence homologous to the mRNA of a target gene and an antisense RNA strand with a sequence complementary to that sequence. Since siRNA can suppress target gene expression, it can be used in gene knockdown methods or gene therapy.

[0084] As used herein, the term "shRNA (short hairpin RNA)" refers to a single-stranded RNA that is divided into a stem portion that forms a double-stranded portion through hydrogen bonds and a loop portion that has a ring shape. It is processed by proteins such as Dicer to be converted into siRNA and can perform the same function as siRNA.

[0085] As used herein, the term "miRNA (microRNA)" refers to 21 to 23 non-coding RNAs that regulate gene expression post-transcriptionally by promoting the degradation of target RNAs or inhibiting their translation.

[0086] As used herein, the term "ribozyme" refers to an RNA molecule with enzyme-like functionality that recognizes a specific base sequence and cleaves it on its own. A ribozyme may be composed of a region that specifically binds to a complementary base sequence of a target messenger RNA strand and a region that cleaves the target RNA.

[0087] Antisense nucleic acids, siRNA, shRNA, miRNA, ribozymes, etc. that complementarily bind to the DNA or mRNA of the above AMPD3 can inhibit essential activities for the biological functions of any other AMPD3 and / or PDK3 ligand, such as transcription, translocation into the cytoplasm, maturation, or translation of the AMPD3 and / or PDL3 ligand.

[0088] According to one specific example, the AMPD3 activity inhibitor may be a pharmaceutical composition comprising at least one selected from the group consisting of compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs) that specifically bind to the protein of AMPD3.

[0089] As used herein, the term “specific” refers to the ability to bind only to a target protein without affecting other proteins within a cell.

[0090] As used herein, the term "peptide" refers to a compound that is a small unit that constitutes a protein and has a structure in which two or more amino acids are linked through peptide bonds.

[0091] As used herein, the term "peptide mimetics" refers to artificial peptide molecules created by mimicking or improving the structure and function of biomolecules. Peptide mimetics include those with structures similar to existing peptide molecules, but with improved stability, bioactivity, and specificity in vivo by modifying or adding certain keywords.

[0092] In this specification, the term "chimeric protein" refers to an artificial recombinant protein that is expressed by linking the genes of one or more other proteins to the protein.

[0093] As used herein, the term "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. The antibody may include a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of the antibody molecule. The functional fragment of the antibody molecule means a fragment that possesses at least an antigen-binding function, and may be, for example, Fab, F(ab'), F(ab')2, or Fv. In addition, the antibody includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. In addition, the antibody can be easily produced using techniques well known in the art.

[0094] As used herein, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristic of being able to bind to a target molecule with high affinity and specificity.

[0095] The term "antibody-drug conjugate (ADC)" used in this specification refers to a substance having a form in which a small molecule drug having a specific pharmacological mechanism is loaded onto an antibody having target specificity via a linker.

[0096] Compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs) that specifically bind to the protein of the above AMPD3 can inhibit the essential activity for the biological function of PDK3.

[0097] In one specific example, the pharmaceutical composition comprising the AMPD3 inhibitor may further comprise or be administered in combination with an inhibitor of the expression or activity of TGFBR3 (TGF-beta receptor 3).

[0098] The term "administration" as used herein means physically introducing a composition to a subject using any of a variety of methods and delivery systems known to those of ordinary skill in the art.

[0099] The term "combination administration" as used herein refers to treating or managing a disease by using two or more drugs or treatment methods simultaneously. The combination administration may be administered simultaneously, concurrently, sequentially, consecutively, alternately, or separately, with the AMPD3 expression or activity inhibitor and the TGFBR3 expression or activity inhibitor.

[0100] Simultaneous administration includes administration at substantially the same time. This form of administration may also be referred to as "concomitant" administration. Concurrent administration includes administering the active agents within the same general period of time, for example, on the same day(s), but not necessarily at the same time. Alternate administration includes administering one agent for a specific period of time, for example, for several days or a week, followed by the other agent(s) for a subsequent period of time, for example, for several days or a week, and then repeating the pattern for one or more cycles. Sequential or sequential administration includes administering one agent for a first period of time (e.g., for several days or a week) using one or more doses, followed by the other agent(s) for a second and / or additional period of time (e.g., for several days or a week) using one or more doses. Overlapping schedules may also be used, which include administering the active agents on different days during the treatment period, but not necessarily in a regular order. For example, variations to these general guidelines may be used depending on the formulation used and the condition of the subject.

[0101] In one specific example, the expression inhibitor may be at least one selected from the group consisting of antisense nucleic acid, siRNA, shRNA, miRNA, and ribozyme that complementarily bind to DNA or mRNA of TGFBR3.

[0102] In one specific example, the active inhibitor may be at least one selected from the group consisting of compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs) that specifically bind to the protein of TGFBR3.

[0103] The definitions of the above neurofibromatosis, inhibitors, antisense nucleic acids, siRNA, shRNA, miRNA, ribozymes, peptides, peptide mimetics, fusion proteins, antibodies, aptamers and antibody-drug conjugates (ADCs) are as described above.

[0104] The pharmaceutical composition of the present invention may, for administration, contain a pharmaceutically acceptable carrier, excipient, or diluent in addition to the inhibitor described above. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0105] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions, according to conventional methods. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc. Such solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the above-mentioned active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. In addition to liquid paraffin for oral administration, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be added to prepare the formulation. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrosol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0106] A pharmaceutical composition according to the present invention can be administered to a subject in need of prevention or treatment of neurofibromatosis, comprising an effective amount of an inhibitor of AMPD3 or its ligand.

[0107] The above effective amount may be a "therapeutically effective amount" or a "prophylactically effective amount." The "therapeutically effective amount" refers to any amount that, when the drug or therapeutic agent is used alone or in combination with other therapeutic agents, can exhibit a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or a prevention of damage or disability due to disease affliction. The "prophylactically effective amount" refers to any amount that inhibits the occurrence or recurrence of cancer in a subject. The level of the above effective amount may be determined based on factors such as the severity of the subject's condition, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0108] The above "subject" includes a human or any non-human animal, wherein the non-human animal may be a vertebrate, such as a primate, dog, cow, horse, pig, rodent, such as a mouse, rat, guinea pig, etc. In the present specification, the "subject" is used interchangeably with "individual" and "patient".

[0109]

[0110] Another aspect provides a kit for preventing or treating neurofibromatosis comprising an AMPD3 inhibitor.

[0111] In one specific example, the kit may further comprise an inhibitor of expression or activity of TGFBR3.

[0112] The definitions of the above neurofibromatosis, inhibitors, antisense nucleic acids, siRNA, shRNA, miRNA, ribozymes, peptides, peptide mimetics, fusion proteins, antibodies, aptamers and antibody-drug conjugates (ADCs) are as described above.

[0113] Another aspect provides the use of an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase) for preventing or treating neurofibromatosis.

[0114] Another aspect provides a method for preventing or treating neurofibromatosis, comprising administering to a subject in need thereof an effective amount of an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase).

[0115] Another aspect provides the use of an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase) for use in the manufacture of a pharmaceutical preparation for the prevention or treatment of neurofibromatosis.

[0116] The terms and methods described for the above inventions apply equally to each invention.

[0117] The present invention relates to a biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent including AMPD3 and a pharmaceutical composition for preventing or treating neurofibromatosis including an AMPD3 inhibitor and its use. The composition according to one aspect is not only highly useful as a useful diagnostic marker for sensitivity to a treatment agent in patients with neurofibromatosis, but also has the advantage of being a new treatment target for neurofibromatosis (NF1).

[0118] Figures 1a and 1b are the results of analyzing gene expression patterns in normal control and NF1 patient cells: Figure 1a shows different expression patterns of genes in normal control and NF1 patient cells through differential expressed gene (DEG) analysis, and Figure 1b is a graph showing genes with increased or decreased expression in normal control and NF1 patient cells.

[0119] Figures 2a and 2b show the results of analyzing the functions of genes or proteins and the biological pathways to which they belong in the normal control group and patients with neurofibromatosis type 1: Figure 2a shows the results of Gene ontology (GO) analysis, and Figure 2b shows the results of KEGG pathway analysis.

[0120] Figure 3 shows the results of confirming samples in which full-length NF1 protein is produced in response to the drug Ataluren and those in which it is not.

[0121] Figure 4 shows the results of measuring ERK activity in samples that responded to the drug Ataluren (NF-09, NF-11) and samples that did not respond (NF-25, NF-31).

[0122] Figure 5 shows the transcriptome of the groups that responded to the drug Ataluren (NF-09, NF-11) and the groups that did not respond (NF-25, NF-31) as a heatmap.

[0123] Figures 6a and 6b are graphs showing the results of confirming mRNA expression before and after ataluren drug treatment in fibroblasts of NF1 patients or normal control groups using reverse transcription-quantitative PCR.

[0124] Figure 7 shows the results of confirming the expression levels of AMPD3 and TGFBR3 proteins in fibroblasts of NF1 patients using western blot.

[0125] Figure 8 shows the results of confirming the expression levels of AMPD3 and TGFBR3 proteins in blood plasma samples from normal controls and NF1 patients using western blot.

[0126] Figure 9 is a photograph showing the results of measuring the expression level of AMPD3 protein in a normal cell line, a pyriform neurofibroma (PN) cell line, and a malignant peripheral nerve sheath tumor (MPNST) cell line.

[0127] Figure 10 is a graph showing the results of measuring the mRNA expression levels of AMPD3 and NF1 in Schwann cell lines treated with AMPD3-specific siRNA and NF1-specific siRNA.

[0128] Figure 11 is a photograph showing the results of Western blotting to determine the AMPD3 protein level and phospho-ERK (p-ERK) protein level in normal Schwann cell lines, plexiform neurofibroma (PN) cell lines, and malignant peripheral nerve sheath tumor (MPNST) cell lines transfected with the siRNAs in Table 2.

[0129] Figure 12 is a graph showing the results of measuring the cell viability of normal Schwann cell lines, plexiform neurofibroma (PN) cell lines, and malignant peripheral nerve sheath tumor (MPNST) cell lines treated with siRNA of the AMPD3 gene.

[0130] Figure 13 is a graph showing the results of measuring apoptosis activity in normal Schwann cell lines, plexiform neurofibroma (PN) cell lines, and malignant peripheral nerve sheath tumor (MPNST) cell lines treated with siRNA of the AMPD3 gene.

[0131] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0132]

[0133] Example 1. Selection of patients with neurofibromatosis who have a stop codon mutation in the NF1 gene.

[0134] DNA extracted from patients with neurofibromatosis (NF) was amplified using polymerase chain reaction (PCR) to identify the NF1 gene, the causative gene of NF1. Sanger sequencing was then performed. Based on the analysis results, 22 patients with a stop codon mutation in the NF1 gene were identified. Information on the NF1 gene stop codon mutations in these patients is provided in Table 1.

[0135] IDMutation of NF1NF-08c.7759G>Tp.Glu2587TerNF-09c.5424C>Tp.Arg1748TerNF-10c.1381C>Tp. Arg461TerNF-11c.2560C>Tp.Gln854TerNF-13c.7039G>Tp.Glu2347TerNF-14c.4537C >Tp.Arg1513TerNF-19c.4084C>Tp.Arg1362TerNF-20c.4243G>Tp.Glu1415TerNF-21 c.3916C>Tp.Arg1306TerNF-22c.1381C>Tp.Arg461TerNF-23c.4537C>Tp.Arg1513Ter NF-24c.3763C>Tp.Gln1255TerNF-25c.6792C>Ap.Tyr2264TerNF-26c.3978T>Gp.Leu 993TerNF-27c.2953C>Tp.Gln985TerNF-28c.1797G>Ap.Trp599TerNF-29c.6792C>Ap. Tyr2264TerNF-30c.3916C>Tp.Arg1306TerNF-31c.3565C>Tp.Gln1189TerNF-32c.19 04delp.Pro635Leufs*53NF-33c.2382T>Ap.Tyr794TerNF-34c.7486C>Tp.Arg2496Ter

[0136]

[0137] Example 2. Transcriptome analysis of normal controls and patients with neurofibromatosis type 1.

[0138] 2.1 Differential expressed gene (DEG) analysis

[0139] Differential expressed gene (DEG) analysis was performed using the transcriptomes of NF-09, NF-11, NF-25, and NF-31 from the normal control group and neurofibromatosis type 1 patients of Example 1. Specifically, the transcriptomes of fibroblasts from the normal control group and NF1 patients were obtained, and transcript assembly was performed using the StringTie program. The expression level of each sample was analyzed as the FPKM (Fragments per kilobase of transcript per million mapped reads) value. After normalization with relative log expression (RLE) for comparison between samples, statistical analysis was performed using the DESeq2 R library.

[0140]

[0141] The above DEG analysis confirmed that genes showed different expression patterns in normal control and NF1 patient cells.

[0142] The results are shown in Figures 1a and 1b.

[0143] Figures 1a and 1b are the results of analyzing gene expression patterns in normal control and NF1 patient cells: Figure 1a shows different expression patterns of genes in normal control and NF1 patient cells through differential expressed gene (DEG) analysis, and Figure 1b is a graph showing genes with increased or decreased expression in normal control and NF1 patient cells.

[0144] In Figure 1a, the color scale from yellow to blue represents log2 (fold change) values ​​from large to small. In Figure 1b, the significance of genes (Differentially Expressed Genes, DEGs) with increased or decreased expression in cells from NF1 patients compared to normal controls is shown on the y-axis versus the fold change on the x-axis.

[0145] As shown in Figures 1a and 1b, it was confirmed that the gene expression patterns of the normal control group and NF1 patients were different, and it was confirmed that 530 genes had increased expression and 243 genes had decreased expression in the cells of NF1 patients compared to the normal control group.

[0146]

[0147] 2.2 Gene ontology (GO) analysis and KEGG pathway analysis

[0148] Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the transcriptomes of NF-09, NF-11, NF-25, and NF-31 from the normal control group and neurofibromatosis type 1 patients of Example 1. Specifically, the Log2 fold change values ​​of NF-09, NF-11, NF-25, and NF-31, which are fibroblasts of each NF1 patient, were compared with the normal control group samples using the method of Example 2.1, and Gene ontology (GO) analysis was performed on the list of statistically significant genes based on the g:Profiler tool (http: / biit.cs.ut.ee / gprofiler / ). In addition, the KEGG pathway was analyzed for the gene list using the DAVID database (https: / david.ncifcrf.gov / summary.jsp), and the results are shown in Figs. 2a and 2b.

[0149] Figures 2a and 2b show the results of analyses of the functions of genes or proteins and the biological pathways to which they belong in the normal control group and patients with neurofibromatosis type 1: Figure 2a shows the results of Gene Ontology (GO) analysis, and Figure 2b shows the results of KEGG pathway analysis. Statistical significance was indicated as *p<0.05, **p<0.01, and ***p<0.001.

[0150] As shown in Figures 2a and 2b, the analysis confirmed that genes involved in the Ras-MAPK signaling pathway, as well as the PI3K-AKT signaling pathway and the Wnt signaling pathway, were differentially expressed in fibroblasts from NF1 patients compared to normal control samples (normal fibroblasts).

[0151]

[0152] Example 3. Measurement of neurofibromatosis treatment response in patients with neurofibromatosis type 1.

[0153] 3.1 Classification by neurofibromatosis treatment response

[0154] Among the skin fibroblasts of patients with neurofibromatosis type 1 in Example 1, samples were divided into two groups: those that produced full-length NF1 protein (Neurofibromin) in response to the drug Ataluren, which reads through the premature stop codon, and those that did not.

[0155] Specifically, samples from skin fibroblasts of normal controls and patients with neurofibromatosis type 1 were extracted before and after Ataluren treatment using RIPA buffer, and the proteins were electrophoresed on an 8% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA. The membrane was incubated with a primary antibody recognizing the C-terminus of neurofibromin, washed, and then incubated with a secondary antibody conjugated to horseradish peroxidase, followed by washing. The protein amount was analyzed using an ECL reagent and a CHEMI-DOC imaging system.

[0156] Figure 3 shows the results of confirming samples in which full-length NF1 protein is produced in response to the drug Ataluren and those in which it is not.

[0157] Samples were divided into those in which the NF1 protein (Neurofibromin) was produced at a level similar to normal by the drug Ataluren (NF-09, NF-11) and those in which there was no drug response (NF-25, NF-31).

[0158]

[0159] 3.2 Confirming the function of restored neurofibromin

[0160] The function of restored Neurofibromin was confirmed by measuring ERK activity in Ataluren drug-responsive (NF-09, NF-11) and non-responsive (NF-25, NF-31) samples.

[0161] Specifically, samples from skin fibroblasts of normal controls and neurofibromatosis type 1 patients before and after Ataluren treatment were extracted using RIPA buffer, and the proteins were electrophoresed on a 10.5% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA. The membrane was incubated with a primary antibody recognizing phospho-ERK1 / 2 (T202 / Y204) or ERK1 / 2, washed, and then incubated with a secondary antibody conjugated to horseradish peroxidase, followed by washing. The protein amount was analyzed using an ECL reagent and a CHEMI-DOC imaging system.

[0162] The results are shown in Fig. 4.

[0163] Figure 4 shows the results of measuring ERK activity in samples that responded to the drug Ataluren (NF-09, NF-11) and samples that did not respond (NF-25, NF-31).

[0164] As shown in Fig. 4, the function of Neurofibromin recovered in samples (NF-09, NF-11) responsive to Ataluren drug was confirmed.

[0165]

[0166] Example 4. Discovery of biomarkers predicting sensitivity to neurofibromatosis treatment 4.1 Selection of potential biomarker candidates showing differences in expression levels due to neurofibromatosis treatment

[0167] A heatmap was created to analyze the transcriptomes in the ataluren-responsive (NF-09, NF-11) and non-responsive (NF-25, NF-31) groups. The heatmap is shown in Fig. 5.

[0168] Figure 5 shows the transcriptome of the groups that responded to the drug Ataluren (NF-09, NF-11) and the groups that did not respond (NF-25, NF-31) as a heatmap.

[0169] By analyzing the heatmap data in Figure 5, we selected candidate groups that showed statistically significant differences in gene expression patterns only in the groups that responded to ataluren drug (NF-09, NF-11).

[0170] The mRNA expression of the selected candidates was confirmed again by reverse transcription-quantitative PCR before and after ataluren drug treatment in NF1 patient or normal control fibroblasts, and the results are shown in Figures 6a and 6b.

[0171] As shown in Figures 6a and 6b, among the candidate groups that showed statistically significant differences in gene expression patterns only in the group responsive to ataluren drug (NF-09, NF-11), AMPD3 and TGFBR3, which are functionally related to neurofibromin (NF1) and secreted into the extracellular matrix and can be detected in the blood (plasma) or urine of patients, were selected as potential biomarker candidates predicting sensitivity to neurofibromatosis treatment.

[0172]

[0173] 4.2 Confirmation of AMPD3 and TGFBR3 protein expression levels in fibroblasts from selected NF1 patients

[0174] The expression levels of AMPD3 and TGFBR3 proteins were confirmed by Western blot in skin fibroblasts from normal individuals and neurofibromatosis patients selected in Example 1 using the following method.

[0175] Specifically, samples from skin fibroblasts of normal controls and neurofibromatosis type 1 patients before and after Ataluren treatment were extracted using RIPA buffer, and the proteins were electrophoresed on a 10.5% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA. The membrane was incubated with a primary antibody recognizing a specific protein (AMPD3 or TGFBR3), washed, and then incubated with a secondary antibody conjugated to horseradish peroxidase, followed by washing. The protein amount was analyzed using an ECL reagent and a CHEMI-DOC imaging system.

[0176] Figure 7 shows the results of confirming the expression levels of AMPD3 and TGFBR3 proteins in fibroblasts of NF1 patients using western blot.

[0177] As shown in Figure 7, it was confirmed that the expression of AMPD3 and TGFBR3 proteins was increased in fibroblasts (NF-09, NF-11, NF-25, NF-31) of NF1 patients compared to the normal control group before drug treatment (control).

[0178] In addition, when comparing the drug-responsive group (NF-09, NF-11) and the non-responsive group (NF-25, NF-31) after drug (Ataluren) treatment, it was confirmed that the expression levels of AMPD3 and TGFBR3 decreased only in the responsive group. In other words, a decrease in AMPD3 and TGFBR3 expression was confirmed as NF1 recovered.

[0179] This means that NF1 treatment monitoring is possible using AMPD3 and TGFBR3 protein expression levels in patient samples.

[0180]

[0181] Example 5. Discovery of a new therapeutic target for neurofibromatosis (NF1).

[0182] 5.1 Confirmation of AMPD3 and TGFBR3 protein expression levels in blood plasma samples from selected NF1 patients

[0183] The expression levels of AMPD3 and TGFBR3 proteins were confirmed by Western blot using the method of Example 4.2 in blood plasma samples from normal individuals and patients with neurofibromatosis type 1.

[0184] Figure 8 shows the results of confirming the expression levels of AMPD3 and TGFBR3 proteins in blood plasma samples from normal controls and NF1 patients using western blot.

[0185] As shown in Figure 8, it was confirmed that the AMPD3 and TGFBR3 protein levels were higher in NF1 patient samples compared to the normal control group.

[0186] This means that it has a high potential for use in patient-specific NF1 treatment monitoring.

[0187]

[0188] 5.2 Confirmation of AMPD3 protein expression in normal cell lines and three neurofibromatosis cell lines

[0189] The expression of AMPD3 was confirmed in four human Schwann cell lines (normal cell line and neurofibroma cell line). Specifically, the expression levels of AMPD3 protein were compared in normal cell line, plexiform neurofibroma (PN) cell line, and malignant peripheral nerve sheath tumor (MPNST) cell line. The normal cell line was ipn02.3 (NF1 + / + ), the neurofibromatosis (PN) cell line ipNF95.6 (NF1 - / - ), the peripheral nerve fiber schwannoma (MPNST) cell line is sNF02.2 (NF1 + / - ) and sNF96.2 (NF1 - / - ) was used. Among the peripheral nerve fiber cell lines, sNF96.2 (NF1 - / - ) is sNF02.2 (NF1 + / -) is a case of severe malignancy. The results of confirming the level of AMPD3 protein expression in the above cell line are shown in Figure 9.

[0190] Figure 9 is a photograph showing the results of measuring the expression level of AMPD3 protein in a normal cell line, a pyriform neurofibroma (PN) cell line, and a malignant peripheral nerve sheath tumor (MPNST) cell line.

[0191] As shown in Figure 9, AMPD3 protein levels were significantly higher in NF1-associated neurofibromatosis cell lines than in normal cell lines. In particular, AMPD3 expression and ERK activity were found to increase as NF1-associated malignancy progressed.

[0192] This indicates that the expression level of AMPD3 was significantly increased in neurofibromatosis (PN) and malignant peripheral nerve sheath tumor (MPNST) cell lines compared to normal cell lines, suggesting that AMPD3 has the potential as a new therapeutic target for NF1-associated neurofibromatosis.

[0193]

[0194] Example 6. Identification of a novel therapeutic target for neurofibromatosis (NF1).

[0195] 6.1 Knockdown of target genes using siRNA

[0196] To evaluate the anticancer effect of AMPD3, AMPD3-specific siRNA and NF1-specific siRNA were designed and knockdown experiments were conducted.

[0197] Specifically, the normal Schwann cell line, plexiform neurofibroma (PN) cell line, and malignant peripheral nerve sheath tumor (MPNST) cell line of Example 4 were seeded in 6-well plates (SPL) and transfected with 50 nM of synthetic siRNA duplex (siAMPD3, siNF1) or siRNA control duplex (Bioneer, Republic of Korea) using Lipofectamine RNAiMax (Invitrogen, USA). The sequences of the synthetic siRNA duplexes are shown in Table 2.

[0198] AMPD3 siRNAsSense strand 5′-GUUCAGCCUUCAUGAGAUGUUAA-3′SEQ ID NO: 1Anti-sense strand 5′-AACAUCUCAUGAAGGCUGAAAU-3′SEQ ID NO: 2NF1 siRNAsSense strand 5′-GAAGGUUGCGCAGUUAGCAGUUA-3′SEQ ID NO: 3Anti-sense strand 5′-ACUGCUAACUGCGCAACCUUAU-3′SEQ ID NO: 4

[0199]

[0200] 6.2 Confirmation of reduced mRNA expression of target genes in cells transfected with siRNA

[0201] To confirm whether the expression of the target genes was successfully knocked down, the relative mRNA expression levels of AMPD3 and NF1 were measured using quantitative PCR in Schwann cell lines transfected with the siRNAs shown in Table 2 above.

[0202] Specifically, cells transfected with the siRNA were collected, RNA was extracted using TRIzol reagent, and cDNA was synthesized using oligo dT(12-18) primer (Invitrogen), random hexamer (Invitrogen), and SuperScript III reverse transcriptase (Invitrogen). Specific genes were amplified and analyzed using the synthesized cDNA and SYBR green reagent using a quantitative PCR system. The relative mRNA expression of the target gene was normalized to the expression of GAPDH.

[0203] Figure 10 is a graph showing the results of measuring the mRNA expression levels of AMPD3 and NF1 in Schwann cell lines treated with AMPD3-specific siRNA and NF1-specific siRNA.

[0204] As shown in Figure 10, it was confirmed that the mRNA expression levels of AMPD3 and NF1 were reduced in the group treated with siRNA of the AMPD3 and NF1 genes compared to the control group that was not treated with siRNA. This means that the siRNA shown in Table 2 effectively inhibits the expression of the AMPD3 and NF1 genes.

[0205]

[0206] Example 7. Confirmation of anticancer effects due to decreased expression of AMPD3 in cells transfected with siRNA.

[0207] 7.1 Confirmation of AMPD3 protein and phospho-ERK (p-ERK) protein levels in neurofibromatosis (PN) cell lines and malignant peripheral nerve sheath tumor (MPNST) cell lines.

[0208] AMPD3 protein levels and phospho-ERK (p-ERK) protein levels were determined by Western blotting in neurofibromatosis (PN) cell lines and malignant peripheral nerve sheath tumor (MPNST) cell lines transfected with siRNA using the method of Example 6.1. Measurement of phospho-ERK (p-ERK) protein levels can confirm the activity of the MEK-ERK signaling pathway.

[0209] Figure 11 is a photograph showing the results of Western blotting to determine the AMPD3 protein level and phospho-ERK (p-ERK) protein level in normal Schwann cell lines, plexiform neurofibroma (PN) cell lines, and malignant peripheral nerve sheath tumor (MPNST) cell lines transfected with the siRNAs in Table 2.

[0210] As shown in Figure 11, it was confirmed that a decrease in AMPD3 protein expression level resulted in a decrease in ERK activity. In addition, when normal cell lines were treated with NF1 siRNA, the AMPD3 protein expression level increased and the p-ERK protein level also increased compared to the control group (siControl treatment).

[0211] This means that when AMPD3 siRNA treatment reduces the amount of AMPD3 protein expression, ERK activity decreases, resulting in an anticancer effect on neurofibromas, and that NF1 and AMPD3 have a negative correlation (anti-correlation).

[0212]

[0213] 7.2 Confirmation of decreased cell viability

[0214] According to the method of Example 6.1, normal Schwann cell line (ipn02.3), plexiform neurofibroma (PN) cell line (ipNF95.6), and malignant peripheral nerve sheath tumor (MPNST) cell lines (NF02.2, and sNF96.2) were treated with siRNA against AMPD3, and 72 hours later, a CCK-8 assay was performed to measure cell survival.

[0215] Specifically, the normal Schwann cell line, plexiform neurofibroma (PN) cell line, and malignant peripheral nerve sheath tumor (MPNST) cell line were seeded in a 96-well plate, and each was treated with AMPD3 siRNA. After 72 hours, 10 μL of CCK-8 assay reagent (Dojindo Molecular Technology, Japan) was added to each well, and the cells were incubated for 3 hours in a 37°C incubator, and then the absorbance at 450 nm was measured using a microplate reader. The cell viability was analyzed by calculating the measured value.

[0216] Figure 12 is a graph showing the results of measuring the cell viability of normal Schwann cell lines, plexiform neurofibroma (PN) cell lines, and malignant peripheral nerve sheath tumor (MPNST) cell lines treated with siRNA of the AMPD3 gene.

[0217] As shown in Figure 12, it was confirmed that the cell viability of the PN cell line and the MPNST cell line treated with siRNA for the AMPD3 gene was reduced compared to the cell viability of the PN cell line and the MPNST cell line not treated with AMPD3 siRNA.

[0218] Specifically, in normal cell lines, there was no change in cell viability depending on the presence or absence of AMPD3 siRNA treatment, but in plexiform neurofibroma (PN) cell lines and malignant peripheral nerve sheath tumor (MPNST) cell lines, it was confirmed that the cell number was reduced by up to 50% when AMPD3 siRNA was treated.

[0219] In addition, when siRNA for the AMPD3 gene was treated, it was confirmed that the cell viability of sNF96.2, a more severe malignant peripheral nerve fiber schwannoma (MPNST) cell line, was further reduced.

[0220] This means that when AMPD3 expression is inhibited by siRNA treatment, a statistically significant anticancer effect is observed in peritoneal neurofibromatosis (PN) cell lines (ipNF95.6) and malignant peripheral nerve sheath tumor (MPNST) cell lines (NF02.2 and sNF96.2).

[0221]

[0222] 7.3 Confirmation of increased apoptosis activity

[0223] Normal Schwann cell line (ipn02.3), plexiform neurofibroma (PN) cell line (ipNF95.6), and malignant peripheral nerve sheath tumor (MPNST) cell lines (NF02.2 and sNF96.2) were treated with siRNA for the AMPD3 gene according to the method of Example 6.1, and 72 hours later, a caspase 3 / 7 assay was performed to confirm the activity of apoptosis.

[0224] Specifically, normal Schwann cells, cystic neurofibroma cells, and MPNST cells were seeded in opaque and white 96-well plates, treated with each siRNA, and 72 hours later, 50 μl of Promega's caspase 3 / 7-glo reagent was added to each well and incubated for 30 minutes. The level of caspase 3 / 7 was measured using a luminometer capable of detecting this fluorescent substance. Apoptotic activity was evaluated by calculating from the measured value.

[0225] Figure 13 is a graph showing the results of measuring apoptosis activity in normal Schwann cell lines, plexiform neurofibroma (PN) cell lines, and malignant peripheral nerve sheath tumor (MPNST) cell lines treated with siRNA of AMPD3.

[0226] As shown in Figure 13, in normal cell lines, there was no change in apoptosis activity depending on the presence or absence of AMPD3 siRNA treatment, but it was confirmed that the apoptosis activity of the plexiform neurofibroma (PN) cell line (ipNF95.6) and malignant peripheral nerve sheath tumor (MPNST) cell lines (sNF02.2, sNF96.2) treated with siRNA for AMPD3 increased compared to the apoptosis activity of the plexiform neurofibroma (PN) cell line and malignant peripheral nerve sheath tumor (MPNST) cell line (control) that were not treated with siRNA.

[0227] In addition, when the above-mentioned malignant peripheral nerve sheath tumor (MPNST) cell line was treated with siRNA for the AMPD3 gene, it was confirmed that apoptosis activity was further increased in the sNF96.2 cell line, which is more malignant among the MPNST cell lines.

[0228] This means that when the expression level of the AMPD3 gene is inhibited by siRNA treatment, a statistically significant anticancer effect is observed in the pyloric neurofibroma (PN) cell line (ipNF95.6) and malignant peripheral nerve sheath tumor (MPNST) cell lines (sNF02.2, sNF96.2). In particular, among the malignant peripheral nerve sheath tumor (MPNST) cell lines, a more effective anticancer effect is observed in the sNF96.2 cell line, which is more malignant.

Claims

1. A biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis type 1 (NF1), comprising a preparation capable of measuring the expression level of the AMPD3 (Adenosine monophosphate deaminase) protein or a gene encoding it.

2. In paragraph 1, A neurofibromatosis treatment agent is a compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof, a biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent: [Chemical Formula 1] In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R is hydrogen or halogen.

3. In paragraph 2, A biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent, wherein the compound represented by the chemical formula 1 above is 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.

4. In paragraph 1, A biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent, wherein the agent capable of measuring the expression level of the above protein is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, and a nanoparticle that specifically bind to the above protein.

5. In paragraph 1, A biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent, wherein the agent capable of measuring the expression level of the gene encoding the above protein is selected from the group consisting of a primer pair, a probe, and an antisense nucleotide that specifically bind to the above gene.

6. In paragraph 1, A biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent, wherein the above neurofibromatosis has a stop codon mutation in the NF1 gene.

7. In paragraph 1, A biomarker composition for predicting sensitivity to a neurofibromatosis treatment agent, wherein the biomarker composition further comprises an inhibitor of expression or activity of TGFBR3 (TGF-beta receptor 3).

8. A kit for predicting sensitivity to a neurofibromatosis treatment agent, comprising a biomarker composition according to any one of claims 1 to 7.

9. A method for providing information necessary for predicting sensitivity to a neurofibromatosis treatment agent, comprising the step of measuring the expression level of AMPD3 protein or a gene encoding it in a biological sample obtained from an individual.

10. In paragraph 9, A method for providing information necessary for predicting sensitivity to a neurofibromatosis treatment agent, wherein the method for providing the above information further comprises a step of measuring the expression level of TGFBR3 protein or a gene encoding it.

11. A pharmaceutical composition for preventing or treating neurofibromatosis containing an inhibitor of the expression or activity of AMPD3 (Adenosine monophosphate deaminase).

12. In claim 11, A pharmaceutical composition, wherein the AMPD3 expression inhibitor is at least one selected from the group consisting of antisense nucleic acid, siRNA, shRNA, miRNA, and ribozyme that complementarily bind to DNA or mRNA of AMPD3.

13. In claim 11, A pharmaceutical composition, wherein the AMPD3 activity inhibitor is at least one selected from the group consisting of compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs) that specifically bind to the AMPD3 protein.

14. In claim 11, A pharmaceutical composition further comprising an inhibitor of expression or activity of TGFBR3 (TGF-beta receptor 3).

15. In claim 14, A pharmaceutical composition, wherein the expression inhibitor is at least one selected from the group consisting of antisense nucleic acid, siRNA, shRNA, miRNA, and ribozyme that complementarily bind to DNA or mRNA of TGFBR3.

16. In claim 14, A pharmaceutical composition, wherein the above-mentioned active inhibitor is at least one selected from the group consisting of compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs) that specifically bind to the protein of TGFBR3.

17. A kit for preventing or treating neurofibromatosis comprising an inhibitor of the expression or activity of AMPD3.

18. In claim 17, A kit for preventing or treating neurofibromatosis, wherein the kit further comprises an inhibitor of expression or activity of TGFBR3.

19. Use of an inhibitor of expression or activity of AMPD3 (Adenosine monophosphate deaminase) for preventing or treating neurofibromatosis.

20. A method for preventing or treating neurofibromatosis, comprising a step of administering an effective amount of an inhibitor of expression or activity of AMPD3 (Adenosine monophosphate deaminase) to a subject in need thereof.

21. Use of an inhibitor of expression or activity of AMPD3 (Adenosine monophosphate deaminase) for use in the manufacture of a pharmaceutical preparation for the prevention or treatment of neurofibromatosis.

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