Application of STAT3 or phosphorylation inhibitor thereof in prevention or treatment of silent corticotropin adenoma

Abstract

The invention relates to an application of STAT3 or a phosphorylation inhibitor thereof in prevention or treatment of silent corticotropin adenoma, and particularly provides an application of STAT3 or a protein phosphorylation inhibitor thereof in prevention and / or treatment of silent corticotropin adenoma. According to the invention, it is found for the first time that a signal transduction and transcriptional activation factor 3 (Signal Transduction and Transcriptional Activator of Of Transcriptional Activator 3, STAT3) and a phosphorylation site of the STAT3 have specific enrichment in silent corticotropin adenoma, and the application of the STAT3 to the silent corticotropin adenoma has the advantages that the specificity of the silent corticotropin adenoma and the phosphorylation site of the silent corticotropin adenoma is improved;

Description

Technical Field

[0001] The present invention relates to the fields of oncology and diagnosis, and more particularly to the use of STAT3 or a phosphorylation inhibitor thereof in preventing or treating silent corticotroph adenomas. Background Art

[0002] Pituitary adenoma, also known as pituitary neuroendocrine tumor (PitNET), originates from the anterior pituitary of the normal pituitary gland. It is the most common neuroendocrine tumor and the second most common intracranial tumor, accounting for 15% of all intracranial tumors.

[0003] Pituitary adenomas can be classified in multiple ways (Table 1). Based on the transcription factor lineage, pituitary adenomas can be divided into four major lineages: PIT1, SF1, TPIT, and NULL. Each lineage has its own pathological and molecular characteristics. Based on hormone secretion, pituitary adenomas can be divided into functional pituitary adenomas and silent pituitary adenomas. The former secretes excessive growth hormone, prolactin, thyroid-stimulating hormone, adrenocorticotropic hormone, etc., leading to systemic endocrine disorders; the latter does not secrete pituitary hormones and mainly compresses surrounding neural tissues (such as the hypothalamus, brainstem, optic nerve, and oculomotor nerve) through mass effect, causing patients to experience symptoms such as visual field defects, decreased vision, decreased consciousness, and even coma. At the same time, they surround or invade surrounding blood vessels (such as the internal carotid artery) and bone (such as the sphenoid sinus and skull base slope) and other important tissues, all of which make surgical resection of the tumor extremely difficult.

[0004] Table 1 Classification of pituitary adenomas

[0005]

[0006]

[0007] Among these subtypes, silent TPIT-lineage tumors, also known as silent corticotroph adenoma (SCA), are the subtype with the worst prognosis among all pituitary adenomas ( Figure 1 Poorly differentiated silent ACTH adenomas have a nearly 50% chance of recurrence within 10 years. This subtype of pituitary adenoma is more common in women, but the risk of recurrence in men after surgical resection is significantly higher than in women.

[0008] Currently, the first-line treatment for silent ACTH adenomas is surgical resection, with radiotherapy being considered for residual tumors or local recurrences. There are currently no effective drugs to control tumors clinically. Upon discovery of tumor recurrence, patients can only control tumor progression through additional surgical resection or radiotherapy. However, surgical resection can be challenging due to the tumor's invasion of bone and / or surrounding vascular envelopment, while local scarring from previous surgery also makes exposure of anatomical structures difficult. Furthermore, radiotherapy is also unable to completely control tumor progression. Patients often fall into a painful cycle of recurrence, additional surgery, and additional recurrence.

[0009] Therefore, there is an urgent need in this field to develop new targets that can effectively control or eliminate silent ACTH adenomas and avoid or reduce the need for repeated surgery or radiotherapy after recurrence in patients with this subtype of tumor. Summary of the Invention

[0010] The purpose of the present invention is to provide a new target for effectively controlling or eliminating silent ACTH adenoma and avoiding or reducing the need for repeated surgery or radiotherapy after recurrence in patients with this subtype of tumor.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] First, for patients who have undergone surgical resection of pituitary adenomas, immunohistochemical staining of the resected tumor can be used to determine the tumor subtype and the expression status of STAT3 and its phosphorylation sites. For patients with silent ACTH-producing adenomas or those with high expression of STAT3 or its phosphorylation sites, treatment with STAT3 and its phosphorylation inhibitors can be considered in cases of residual tumor or postoperative recurrence.

[0013] Secondly, for patients who have not undergone surgical resection, whose imaging suggests the possibility of a non-functioning pituitary adenoma, and who do not have the desire or conditions for surgery, considering epidemiological factors (such as non-functioning pituitary tumors in female patients) and imaging features, if the possibility of a silent ACTH adenoma is high, STAT3 and its phosphorylation inhibitors can be used for empirical treatment to control tumor progression or to prepare for subsequent surgical treatment.

[0014] In a first aspect, the present invention provides the use of an inhibitor of STAT3 or its protein phosphorylation for preventing and / or treating silent ACTH adenoma.

[0015] In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, gene editors, or a combination thereof.

[0016] In another preferred embodiment, the inhibitors include Napabucasin / BBI608 and STAT3-IN-1.

[0017] In another preferred embodiment, the inhibitor includes an antibody or antibody fragment that inhibits STAT3 or protein phosphorylation thereof.

[0018] In another preferred embodiment, the STAT3 is derived from mammals; preferably, from humans, mice, rats, or rabbits; more preferably, from humans.

[0019] In another preferred embodiment, the STAT3 protein includes wild-type STAT3 protein and mutant STAT3 protein.

[0020] In another preferred embodiment, the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (ie, the function is the same or substantially the same as that of the wild-type encoded protein).

[0021] In another preferred embodiment, the STAT3 protein includes an active fragment of STAT3 or a derivative thereof.

[0022] In another preferred embodiment, the active fragment or derivative thereof has a homology with STAT3 of at least 90%, preferably 95%, more preferably 98% or 99%.

[0023] In another preferred embodiment, the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, or 100% of the STAT3 activity.

[0024] In another preferred embodiment, the accession number of the STAT3 protein is P40763 (UniProtKB, human STAT3) or P42227 (UniProtKB, mouse STAT3).

[0025] In another preferred embodiment, the inhibitor inhibits the activity and / or expression of STAT3 protein.

[0026] In another preferred embodiment, the inhibitor inhibits the enrichment of STAT3 protein or its phosphorylation sites.

[0027] In another preferred example, the phosphorylation sites of the STAT3 protein include serine at position 726 of the STAT3 protein, and / or serine at position 727 of the STAT3 protein, and / or threonine at position 713 of the STAT3 protein, and / or threonine at position 714 of the STAT3 protein, and / or serine at position 691 of the STAT3 protein, and / or tyrosine at position 704 of the STAT3 protein, and / or tyrosine at position 705 of the STAT3 protein.

[0028] In another preferred example, the STAT3 protein phosphorylation includes phosphorylation of serine at position 726, and / or phosphorylation of serine at position 727, and / or phosphorylation of threonine at position 713, and / or phosphorylation of threonine at position 714, and / or phosphorylation of serine at position 691, and / or phosphorylation of tyrosine at position 704, and / or phosphorylation of tyrosine at position 705.

[0029] In another preferred embodiment, the concentration of the STAT3 protein inhibitor is 4-80 mg / kg body weight, preferably 5-600 mg / kg body weight, and more preferably 25-60 mg / kg body weight.

[0030] In another preferred embodiment, the concentration of the STAT3 protein inhibitor is 0.002-200 μM, preferably, 0.008-150 μM, more preferably, 0.01-100 μM, more preferably, 0.01-50 μM.

[0031] A second aspect of the present invention provides a pharmaceutical composition comprising:

[0032] (a) an inhibitor of STAT3 or its protein phosphorylation; and

[0033] (b) a pharmaceutically acceptable carrier.

[0034] In another preferred embodiment, the pharmaceutical composition further comprises:

[0035] (c) Other drugs for the prevention and / or treatment of silent ACTH adenomas.

[0036] In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, gene editors, or a combination thereof.

[0037] In another preferred embodiment, the inhibitors include Napabucasin / BBI608 and STAT3-IN-1.

[0038] In another preferred embodiment, the inhibitor includes an antibody or a fragment thereof that inhibits STAT3 or protein phosphorylation.

[0039] In another preferred embodiment, in the pharmaceutical composition, the content of component (a) is 1%-99%, preferably, 10%-90%, and more preferably, 30%-70%.

[0040] In another preferred embodiment, in the pharmaceutical composition, the content of component (c) is 1%-99%, preferably, 10%-90%, and more preferably, 30%-70%.

[0041] In another preferred embodiment, in the pharmaceutical composition, the component (a) and the optional component (c) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the pharmaceutical composition.

[0042] In another preferred embodiment, the other drugs for preventing and / or treating silent ACTH adenoma include temozolomide.

[0043] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection form and an oral dosage form.

[0044] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0045] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0046] A third aspect of the present invention provides a medicine kit comprising:

[0047] (a1) A first container, and an inhibitor of STAT3 or its protein phosphorylation, or a drug containing an inhibitor of STAT3 or its protein phosphorylation, located in the first container.

[0048] In another preferred embodiment, the medicine kit further comprises:

[0049] (b1) a second container, and other drugs for preventing and / or treating silent ACTH adenomas located in the second container, or a drug containing other drugs for preventing and / or treating silent ACTH adenomas.

[0050] In another preferred embodiment, the drug kit further comprises (c1) a third container, and a detection reagent for STAT3 or protein phosphorylation thereof located in the third container.

[0051] In another preferred embodiment, the first container, the second container and the third container are the same or different containers.

[0052] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing an inhibitor of STAT3 or its protein phosphorylation.

[0053] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing other medicines for preventing and / or treating silent ACTH adenoma.

[0054] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0055] In another preferred embodiment, the kit further contains instructions.

[0056] In another preferred embodiment, the description records one or more instructions selected from the following group:

[0057] (a) using an inhibitor of STAT3 or its protein phosphorylation for preventing and / or treating silent corticotroph adenomas;

[0058] (b) combining an inhibitor of STAT3 or its protein phosphorylation with other drugs for preventing and / or treating silent ACTH adenomas to prevent and / or treat silent ACTH adenomas;

[0059] (c) detecting the expression level or enrichment level of STAT3 or its protein phosphorylation in patients with silent ACTH adenoma, and administering an inhibitor of STAT3 or its protein phosphorylation to prevent and / or treat silent ACTH adenoma;

[0060] (d) detecting the expression level or enrichment level of STAT3 or its protein phosphorylation in patients with silent ACTH adenoma, and simultaneously administering an inhibitor of STAT3 or its protein phosphorylation; and other drugs for preventing and / or treating silent ACTH adenoma to prevent and / or treat silent ACTH adenoma.

[0061] The fourth aspect of the present invention provides a use of the pharmaceutical composition according to the second aspect of the present invention or the medicine kit according to the third aspect of the present invention for preventing and / or treating silent ACTH adenoma.

[0062] In another preferred embodiment, in the pharmaceutical composition, the effective concentration of the inhibitor of STAT3 or its protein phosphorylation is 4-80 mg / kg body weight, preferably, 5-600 mg / kg body weight, more preferably, 25-60 mg / kg body weight.

[0063] In another preferred embodiment, the concentration of the STAT3 protein or its protein phosphorylation inhibitor is 0.002-200 μM, preferably, 0.008-150 μM, more preferably, 0.01-100 μM, and even more preferably, 0.01-50 μM.

[0064] In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of STAT3 or its protein phosphorylation; and (b) optionally other drugs for preventing and / or treating silent corticotropin adenoma; and (d) a pharmaceutically acceptable carrier.

[0065] In another preferred embodiment, in the pharmaceutical composition or kit, the inhibitor of STAT3 or its protein phosphorylation; and (b) optional other drugs for preventing and / or treating silent adrenocorticotroph adenoma account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the pharmaceutical composition or kit.

[0066] A fifth aspect of the present invention provides a method for preventing and / or treating silent ACTH adenoma, comprising:

[0067] Administering an inhibitor of STAT3 or its protein phosphorylation to a subject in need thereof; or the pharmaceutical composition according to the second aspect of the present invention or the drug kit according to the third aspect of the present invention.

[0068] In another preferred embodiment, the subject includes a human or non-human mammal suffering from silent ACTH adenoma.

[0069] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0070] In another preferred embodiment, the dosage of the STAT3 or protein phosphorylation inhibitor is 4-80 mg / kg body weight, preferably 5-600 mg / kg body weight, more preferably 25-60 mg / kg body weight.

[0071] In another preferred embodiment, the inhibitor of STAT3 or protein phosphorylation thereof is administered once every 2-14 days, preferably once every 7-10 days.

[0072] In another preferred embodiment, the inhibitor of STAT3 or its protein phosphorylation is administered simultaneously or sequentially with other optional drugs for preventing and / or treating silent ACTH adenoma.

[0073] A sixth aspect of the present invention provides a method for screening candidate substances for preventing and / or treating silent ACTH adenomas, the method comprising the steps of:

[0074] (a) In a test group, a test compound is added to the cell culture system, and the expression level (E1) and / or activity (A1) of STAT3 protein in the cells of the test group is observed; and / or the enrichment level (F1) of STAT3 protein or STAT3 protein phosphorylation; in a control group, no test compound is added to the same cell culture system, and the expression level (E0) and / or activity (A0) of STAT3 protein in the cells of the control group is observed; and / or the enrichment level (F0) of STAT3 protein or STAT3 protein phosphorylation;

[0075] Among them, if the expression level (E1) and / or activity (A1) of STAT3 protein in the cells of the test group; and / or the enrichment level (F1) of STAT3 protein or STAT3 protein phosphorylation is significantly lower than that of the control group, it indicates that the test compound is a candidate substance for preventing and / or treating silent adrenocorticotroph adenoma that has an inhibitory effect on the expression and / or activity of STAT3 or an inhibitory effect on the phosphorylation of STAT3 protein.

[0076] In another preferred embodiment, the expression level of STAT3 is obtained by fluorescence quantitative PCR or immunohistochemistry.

[0077] In another preferred embodiment, the enrichment level of the STAT3 protein or the phosphorylation of the STAT3 protein is obtained by immunohistochemistry and / or proteomics / phosphoproteomics detection.

[0078] In another preferred embodiment, the method further comprises the steps of:

[0079] (b) For the candidate substance obtained in step (a), further testing its inhibitory effect on silent ACTH adenoma; and / or further testing whether it has a down-regulating effect on STAT3 protein.

[0080] In another preferred embodiment, the method comprises step (c): administering the candidate substance determined in step (a) to a mammalian model to determine its effect on silent ACTH adenoma in the mammal.

[0081] In another preferred embodiment, the mammal is a mammal suffering from silent ACTH adenoma.

[0082] In another preferred embodiment, the “significantly lower than” means E1 / E0≤1 / 2, preferably ≤1 / 3, more preferably ≤1 / 4.

[0083] In another preferred embodiment, the “significantly lower than” means A1 / A0≤1 / 2, preferably ≤1 / 3, more preferably ≤1 / 4.

[0084] In another preferred embodiment, the “significantly lower than” means F1 / F0≤1 / 2, preferably ≤1 / 3, more preferably ≤1 / 4.

[0085] In another preferred embodiment, the cells include eukaryotic cells.

[0086] In another preferred embodiment, the cells include mammalian cells.

[0087] In another preferred embodiment, the cells include silent ACTH-producing adenoma cells.

[0088] In another preferred embodiment, the cells are cells cultured in vitro.

[0089] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0090] In another preferred embodiment, the candidate substance is selected from the following group: antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, gene editors, or a combination thereof.

[0091] In another preferred embodiment, the candidate substances include antibodies and small molecule compounds.

[0092] In another preferred embodiment, the candidate compound includes an antibody or antibody fragment specific for STAT3 or STAT3 phosphorylation.

[0093] In another preferred embodiment, the candidate substance includes a peptide inhibitor specific for STAT3 or STAT3 phosphorylation.

[0094] In another preferred embodiment, the candidate substances include Napabucasin / BBI608 and STAT3-IN-1.

[0095] A seventh aspect of the present invention provides a method for screening candidate substances for preventing and / or treating silent ACTH adenomas, the method comprising the steps of:

[0096] (i) contacting a candidate substance with a STAT3 protein; and

[0097] (ii) determining the functional effect of the candidate substance on the STAT3 protein, wherein the functional effect indicates that the compound prevents and / or treats silent ACTH adenoma.

[0098] In another preferred embodiment, the STAT3 protein includes recombinant STAT3 protein.

[0099] In another preferred embodiment, the functional effect is determined in vitro.

[0100] In another preferred embodiment, the functional effect is determined by measuring the expression of STAT3.

[0101] In another preferred embodiment, the functional effect is determined by measuring the enrichment level of STAT3 protein or STAT3 protein phosphorylation.

[0102] In another preferred embodiment, the expression level of STAT3 is obtained by fluorescence quantitative PCR or immunohistochemistry.

[0103] In another preferred embodiment, the enrichment level of the STAT3 protein or the phosphorylation of the STAT3 protein is obtained by immunohistochemistry and / or proteomics / phosphoproteomics detection.

[0104] In another preferred embodiment, the functional effect is determined in eukaryotic cells.

[0105] In another preferred embodiment, the eukaryotic cells include mammalian cells.

[0106] In another preferred embodiment, the eukaryotic cells include silent ACTH-producing adenoma cells.

[0107] In another preferred embodiment, the functional effect is determined by measuring the phosphorylation of STAT3.

[0108] In another preferred embodiment, the phosphorylation includes at serine at position 726 of STAT3, and / or at serine at position 727 of STAT3, and / or at threonine at position 713 of STAT3, and / or at threonine at position 714 of STAT3, and / or at serine at position 691 of STAT3, and / or at tyrosine at position 704 of STAT3, and / or at tyrosine at position 705 of STAT3.

[0109] In another preferred embodiment, the functional effect is inhibiting the expression or activity of STAT3; and / or inhibiting the phosphorylation of STAT3.

[0110] In another preferred embodiment, the candidate substances include: antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, and gene editors.

[0111] In another preferred embodiment, the candidate substance includes an antibody or antibody fragment specific for STAT3 or STAT3 phosphorylation.

[0112] In another preferred embodiment, the candidate substance includes an antibody or antibody fragment specific for STAT3 or STAT3 phosphorylation.

[0113] In another preferred embodiment, the candidate substance includes a peptide inhibitor specific for STAT3 or STAT3 phosphorylation.

[0114] In another preferred embodiment, the candidate substance includes an antisense molecule specific for STAT3.

[0115] In another preferred embodiment, the candidate substance includes an RNAi molecule specific for STAT3.

[0116] In another preferred embodiment, the candidate substances include Napabucasin / BBI608 and STAT3-IN-1.

[0117] In an eighth aspect, the present invention provides a use of STAT3 or its protein phosphorylation site, or a detection reagent thereof, (i) as a marker for detecting or diagnosing silent ACTH adenoma; and / or (ii) for preparing a diagnostic reagent or kit for detecting or diagnosing silent ACTH adenoma.

[0118] In another preferred embodiment, the diagnostic reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

[0119] In another preferred embodiment, the protein includes a full-length protein or a protein fragment.

[0120] In another preferred embodiment, the STAT3 is derived from mammals, preferably rodents (such as mice, rats), primates and humans, and more preferably, from patients diagnosed with silent ACTH adenoma.

[0121] In another preferred embodiment, the STAT3 is derived from a patient with silent ACTH adenoma.

[0122] In another preferred embodiment, the accession number of the STAT3 protein is P40763 (UniProtKB, human STAT3) or P42227 (UniProtKB, mouse Stat3).

[0123] In another preferred embodiment, the detection is tissue sample detection, blood sample detection and / or serum sample detection.

[0124] In another preferred embodiment, the detection includes immunohistochemistry and / or proteomics / phosphoproteomics, immunoblotting and fluorescence quantitative PCR detection.

[0125] In another preferred embodiment, the detection includes measuring silent adrenocorticotroph adenoma tissue, other subtypes of pituitary tumors (including but not limited to prolactin cell tumors, somatotropin cell tumors, thyrotropin cell tumors, silent PIT1 lineage tumors, gonadotropin cell tumors, silent SF1 lineage tumors and null cell tumors) or normal pituitary tissue samples.

[0126] In another preferred embodiment, the general ACTH tissue includes paracancerous tissue.

[0127] In another preferred embodiment, the detection reagent includes a specific antibody for STAT3 or its phosphorylation site, a specific binding molecule for STAT3 or its phosphorylation site, a specific amplification primer, a probe or a chip.

[0128] In another preferred embodiment, the STAT3 or its phosphorylation site or its specific antibody or specific binding molecule is coupled with or carries a detectable label.

[0129] In another preferred embodiment, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0130] In another preferred embodiment, the specific antibody against STAT3 or its phosphorylation site is a monoclonal antibody or a polyclonal antibody.

[0131] In another preferred embodiment, the phosphorylation sites include serine at position 726 of STAT3, and / or serine at position 727 of STAT3, and / or threonine at position 713 of STAT3, and / or threonine at position 714 of STAT3, and / or serine at position 691 of STAT3, and / or tyrosine at position 704 of STAT3, and / or tyrosine at position 705 of STAT3.

[0132] A ninth aspect of the present invention provides a diagnostic kit for detecting or diagnosing silent ACTH adenoma, the kit comprising a container containing a detection reagent for detecting STAT3 or its protein phosphorylation site; and a label or instructions indicating that the kit is used for detecting or diagnosing silent ACTH adenoma.

[0133] In another preferred embodiment, the judgment includes prejudgment (prediction).

[0134] In another preferred embodiment, the detection reagent for STAT3 or its protein phosphorylation site comprises:

[0135] (a) Anti-STAT3 or its protein phosphorylation site specific antibodies; and / or

[0136] (b). Specific primers for specifically amplifying mRNA or cDNA of STAT3 or its protein phosphorylation sites.

[0137] In another preferred embodiment, the detection is tissue sample detection, blood sample detection and / or serum sample detection.

[0138] A tenth aspect of the present invention provides a method for detecting or diagnosing silent ACTH adenoma, the method comprising:

[0139] a) providing a test sample from a subject;

[0140] b) detecting the level of STAT3 protein or polynucleotide thereof in the test sample; and

[0141] c) comparing the level of STAT3 determined in step b) with the level of STAT3 in a normal biological sample, thereby detecting or diagnosing silent ACTH adenoma.

[0142] In another preferred embodiment, the subject is a human or non-human mammal.

[0143] In another preferred embodiment, the test sample is silent ACTH-producing adenoma cells or tissues.

[0144] In another preferred embodiment, the level of phosphorylated STAT3 protein is detected.

[0145] In another preferred embodiment, the phosphorylation includes at serine at position 726 of STAT3, and / or at serine at position 727 of STAT3, and / or at threonine at position 713 of STAT3, and / or at threonine at position 714 of STAT3, and / or at serine at position 691 of STAT3, and / or at tyrosine at position 704 of STAT3, and / or at tyrosine at position 705 of STAT3.

[0146] In another preferred embodiment, the detection includes fluorescent quantitative PCR or immunohistochemistry detection, immunohistochemistry and (or) proteomics / phosphoproteomics detection.

[0147] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0148] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 Representative magnetic resonance imaging images of a silent ACTH adenoma are shown. The red dashed line outlines the tumor. The first and second left images are coronal sections, showing the internal carotid artery surrounded by the tumor; the first and second right images are sagittal sections.

[0150] Figure 2 Multi-omics data showed that STAT3 mRNA, protein and its protein phosphorylation sites were enriched in silent ACTH adenomas.

[0151] Figure 3Immunohistochemical staining data showed that STAT3 and its protein phosphorylation sites were enriched in silent ACTH.

[0152] Figure 4 The results showed that the generation of primary tumor organoids from three silent corticotroph adenomas and the screening of STAT3 phosphorylation inhibitors achieved inhibition rates exceeding 99%, suggesting that inhibition of STAT3 phosphorylation sites can significantly inhibit the activity of organoids from this subtype of tumor.

[0153] Figure 5 The results show the use of solvent, STAT3 inhibitor 1 (Napabucasin), and STAT3 inhibitor 2 (STAT3-IN-1) in a primary organoid tumor model. Both STAT3 inhibitors significantly inhibited tumor growth in mice. Six NOD-SCID tumor-bearing mice were included in each group. Significant differences between groups are marked (ns, not significant); *, p < 0.05; **, p < 0.01; ***, p < 0.001). DETAILED DESCRIPTION

[0154] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) or its phosphorylation sites are specifically enriched in silent ACTH adenomas. Furthermore, the inventors unexpectedly discovered that STAT3 or its phosphorylation inhibitors can effectively inhibit silent ACTH adenomas. Based on this, the inventors completed the present invention.

[0155] As described herein, the structural formula of "Napabucasin / BBI608" is shown below:

[0156]

[0157] As described herein, the structural formula of "STAT3-IN-1" is shown below:

[0158]

[0159] Silent ACTH-producing adenoma

[0160] Pituitary adenoma, also known as pituitary neuroendocrine tumor (PitNET), originates from the anterior pituitary of the normal pituitary gland. It is the most common neuroendocrine tumor and the second most common intracranial tumor, accounting for 15% of all intracranial tumors.

[0161] Pituitary adenomas can be classified in multiple ways (Table 1). Based on the transcription factor lineage, pituitary adenomas can be divided into four major lineages: PIT1, SF1, TPIT, and NULL. Each lineage has its own pathological and molecular characteristics. Based on hormone secretion, pituitary adenomas can be divided into functional pituitary adenomas and silent pituitary adenomas. The former secretes excessive growth hormone, prolactin, thyroid-stimulating hormone, adrenocorticotropic hormone, etc., leading to systemic endocrine disorders; the latter does not secrete pituitary hormones and mainly compresses surrounding neural tissues (such as the hypothalamus, brainstem, optic nerve, and oculomotor nerve) through mass effect, causing patients to experience symptoms such as visual field defects, decreased vision, decreased consciousness, and even coma. At the same time, they surround or invade surrounding blood vessels (such as the internal carotid artery) and bone (such as the sphenoid sinus and skull base slope) and other important tissues, all of which make surgical resection of the tumor extremely difficult.

[0162] Table 1 Classification of pituitary adenomas

[0163]

[0164] Among these subtypes, silent corticotroph adenoma (SCA) is the subtype with the worst prognosis among all pituitary adenomas ( Figure 1 Poorly differentiated silent ACTH adenomas have a nearly 50% chance of recurrence within 10 years. This subtype of pituitary adenoma is more common in women, but the risk of recurrence in men after surgical resection is significantly higher than in women.

[0165] sample

[0166] As used herein, the terms "sample" or "specimen" refer to a material that is specifically associated with a subject, from which specific information about the subject can be determined, calculated, or inferred. A sample can consist entirely or in part of biological material from the subject. A sample can also be a material that has been in contact with the subject in a manner that allows testing of the sample to provide information about the subject. A sample can also be a material that has been in contact with other materials that were not from the subject, but that enable the first material to be subsequently tested to determine information about the subject, for example, a sample can be a cleaning solution from a probe or scalpel. A sample can be a source of biological material other than that in contact with the subject, as long as a person skilled in the art can still determine information about the subject from the sample.

[0167] "Biological samples" include tissue sections, such as biopsy samples (e.g., neuronal biopsies) or frozen sections for histological purposes. Such samples also include blood and blood components (e.g., serum, platelets, red blood cells, etc.), cerebrospinal fluid, cultured cells, such as primary cultures, explants, and transformed cells, feces, urine, etc. Biological samples are typically obtained from mammals, such as primates or humans.

[0168] As used herein, "normal biological sample" or "normal individual" refers to a baseline value or control. "Normal" refers to the level of a polynucleotide or nucleic acid in a healthy individual who does not have or will not develop a silent ACTH adenoma (as determined by standard methods, such as immunohistochemistry and / or proteomics / phosphoproteomics testing or using biological samples, such as other subtypes of pituitary tumors (including but not limited to prolactinomas, somatotroph tumors, thyrotropin tumors, silent PIT1 lineage tumors, gonadotroph tumors, silent SF1 lineage tumors and null cell tumors) and normal pituitary tissue).

[0169] Express

[0170] As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or a gene or gene portion, and also includes the appearance of a detection substance associated with expression. For example, cDNA, the binding of a binding ligand (such as an antibody) to a gene or other oligonucleotide, protein or protein fragment, and the chromogenic portion of the binding ligand are all included within the scope of the term "expression." Thus, an increase in half-dot density on an immunoblot such as a western blot also falls within the scope of the term "expression" based on biological molecules.

[0171] The terms "overexpression," "overexpression," or "overexpressed" refer interchangeably to a protein or nucleic acid (RNA) that is transcribed or translated at a detectably higher level in a cell relative to a normal cell. The term includes overexpression due to transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelles, cytoplasm, nucleus, cell surface), and RNA and protein stability (relative to normal cells). Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or conventional techniques for detecting protein (i.e., ELISA, immunohistochemistry). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than normal cells. In some instances, overexpression is a transcription or translation level that is 1-fold, 2-fold, 3-fold, 4-fold or more higher relative to normal cells.

[0172] The terms "underexpress," "underexpression," or "underexpressed" refer interchangeably to proteins or nucleic acids that are transcribed or translated at a detectably lower level in a cell relative to a normal cell. The term includes underexpression due to transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelles, cytoplasm, nucleus, cell surface), and RNA and protein stability (relative to a control). Underexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or conventional techniques for detecting protein (i.e., ELISA, immunohistochemistry). Underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less relative to a control. In some instances, underexpression is a transcription or translation level that is 1-fold, 2-fold, 3-fold, 4-fold or less relative to a control.

[0173] The terms "differentially expressed" or "differentially regulated" generally refer to a protein or nucleic acid that is overexpressed (upregulated) or underexpressed (downregulated) in one sample relative to at least one other sample. For the purposes of the present invention, samples from individuals suspected of having silent ACTH adenomas are typically compared to samples from individuals known to have the condition (positive control) or known to be negative for the condition (negative control).

[0174] STAT3 protein

[0175] As used herein, "STAT3" refers to a member of the signal transducer and activator of transcription (STAT) protein family.

[0176] The protein encoded by the STAT3 gene belongs to the STAT protein family. Under the action of cytokines and growth factors, STAT family members are phosphorylated by receptor-associated kinases, then form homo- or heterodimers and are transported to the cell nucleus, where they function as transcriptional activators. STAT3 protein is activated by phosphorylation in response to various cytokines and growth factors (including IFN, EGF, IL5, IL6, HGF, LIF, and BMP2). STAT3 protein mediates the expression of multiple genes in response to cellular stimulation and therefore plays a key role in many cellular processes, such as cell growth and apoptosis. Studies have shown that the small GTPase Rac1 binds to the STAT3 protein and regulates its activity. The PIAS3 protein is a specific inhibitor of the STAT3 protein. This gene also plays a role in regulating the host response to viral and bacterial infections.

[0177] STAT3 also refers to species homologs, splice variants, polymorphic variants, and conservatively modified variants of STAT3 that have STAT3 signaling activity.

[0178] In the present invention, the terms "protein of the present invention," "STAT3 protein," and "STAT3 polypeptide" are used interchangeably to refer to proteins or polypeptides having the STAT3 amino acid sequence. These include STAT3 proteins with or without an initiator methionine. Furthermore, the term also encompasses full-length STAT3 and fragments thereof. The STAT3 protein referred to herein includes its complete amino acid sequence, its secreted protein, its mutants, and its functionally active fragments.

[0179] The human STAT3 protein has a full length of 770 amino acids (Accession No. P40763 (UniProtKB)). The mouse STAT3 protein has a full length of 770 amino acids (Accession No. P42227 (UniProtKB)).

[0180] In the present invention, the terms "STAT3 gene" and "STAT3 polynucleotide" are used interchangeably and both refer to nucleic acid sequences having a STAT3 nucleotide sequence.

[0181] The genomic length of the human STAT3 gene is 75171 bp (NCBI GenBank accession number is NG_007370 (NCBI, Nucleotide)), and the mRNA sequence of its transcription product is 4899 bp (NCBI GenBank accession number is NM_001369512.1 (NCBI, Nucleotide)).

[0182] The full-length genomic sequence of the mouse STAT3 gene is 83,848 bp (NCBI GenBank accession number AF246978.2 (NCBI, Nucleotide)), and the full-length mRNA sequence of its transcription product is 4,470 bp (NCBI GenBank accession number NM_011486 (NCBI, Nucleotide)).

[0183] Human and mouse STAT3 share 99.87% protein sequence similarity. It should be understood that nucleotide substitutions within codons are acceptable when encoding the same amino acid. Furthermore, it should be understood that nucleotide substitutions are also acceptable when resulting in conservative amino acid substitutions.

[0184] When an amino acid fragment of STAT3 is obtained, a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombinant methods or artificial synthesis methods. For PCR amplification, primers can be designed based on the STAT3 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified in each amplification are spliced ​​together in the correct order.

[0185] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0186] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0187] Currently, DNA sequences encoding proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely through chemical synthesis, which can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0188] The polynucleotide sequences of the present invention can be used to express or produce recombinant STAT3 polypeptides using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0189] (1) Transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the human STAT3 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0190] (2) Host cells cultured in a suitable culture medium;

[0191] (3) Isolate and purify proteins from culture medium or cells.

[0192] In the present invention, the STAT3 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and translation control elements.

[0193] Methods well known to those skilled in the art can be used to construct expression vectors containing a STAT3 encoding DNA sequence and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0194] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0195] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0196] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces bacteria cells; fungal cells, such as yeast; plant cells; insect cells; animal cells, etc.

[0197] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0198] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0199] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0200] The term "recombinant" when used to refer to, for example, a cell, nucleic acid, protein, or vector, represents a cell, nucleic acid, protein, or vector that has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that is derived from a cell that has been so modified. Thus, for example, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell, or expresses native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0201] Specific antibodies

[0202] In the present invention, the terms "antibody of the present invention" and "antibody specific for STAT3 and its phosphorylation sites" are used interchangeably.

[0203] The present invention also includes polyclonal antibodies and monoclonal antibodies specific for human STAT3 polypeptides, particularly monoclonal antibodies. Here, "specificity" refers to the ability of the antibody to bind to the human STAT3 gene product or fragment. Preferably, it refers to antibodies that can bind to the human STAT3 gene product or fragment but do not recognize and bind to other non-related antigenic molecules. The antibodies of the present invention include those that can bind to and inhibit the human STAT3 protein, as well as those that do not affect the function of the human STAT3 protein. The present invention also includes antibodies that can bind to modified or unmodified forms of the human STAT3 gene product.

[0204] The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a mouse antibody but still retain an antibody portion from a human.

[0205] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human STAT3 gene products or antigenic fragments thereof can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human STAT3 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibodies of the present invention can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol. 6:511,1976; Kohler et al., Eur.J.Immunol .6:292,1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies that can block the function of human STAT3 protein and antibodies that do not affect the function of human STAT3 protein. The various antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of the human STAT3 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a peptide synthesizer. Antibodies that bind to unmodified forms of human STAT3 gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing animals with gene products produced in eukaryotic cells (e.g., yeast or insect cells).

[0206] Antibodies against human STAT3 protein can be used in immunohistochemistry to detect human STAT3 protein in specimens (especially tissue samples or serum samples). Because STAT3 protein has an extracellular domain, when the extracellular domain is shed and enters the blood, these soluble STAT3 extracellular domains can become targets for serum detection.

[0207] Generally speaking, antibodies include antibody fragments that retain specificity for an antigen or epitope (e.g., STAT3, phosphorylated STAT3). Antibody inhibitors typically target a functional domain of the antigen, e.g., the dimerization domain of STAT3.

[0208] In the context of assays for compounds that inhibit or treat silent ACTH adenomas or for silent ACTH adenomas markers, the term "functional effect" includes assays for parameters that are indirectly or directly affected by the biomarkers of the invention, such as chemical or phenotypic effects. Thus, functional effects include immediate signaling events, such as transcriptional activation or phosphorylation, or more indirect effects, such as gene expression or apoptosis.

[0209] "Functional effect" includes in vitro, in vivo and ex vivo activities.

[0210] "Assessing a functional effect" means measuring compounds that increase or decrease parameters that are indirectly or directly affected by the biomarkers of the invention, such as physical and chemical or phenotypic effects. Such functional effects can be measured by any method known to those skilled in the art, such as by changes in spectrophotometric properties (e.g., fluorescence, absorption, refractive index); fluid dynamics (e.g., shape), chromatography; or solubility properties of proteins; measuring inducible markers or transcriptional activation of markers; measuring changes in enzyme (e.g., kinase) activity; increasing or decreasing the ability to cause cell proliferation, apoptosis, or cell cycle arrest; measuring changes in cell surface markers. Functional effects can be assessed by many methods known to those skilled in the art, such as microscopy for quantitative or qualitative determination of changes in morphological characteristics, measuring changes in RNA or protein levels of other genes expressed in the cell, measuring RNA stability, identifying downstream or reporter gene expression (CAT, luciferase, β-gal, GFP, etc.), such as by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, etc.

[0211] The "activity" of a silent ACTH adenoma biomarker polypeptide refers to the structural, regulatory, or biochemical function of the polypeptide in its native cells or tissues. The activity of a silent ACTH adenoma biomarker includes both direct and indirect activities. Direct activities of phosphorylated STAT3 include dimerization and transcriptional activation. Exemplary indirect activities are phenotypic changes or responses of cells or tissues resulting from the direct activity of the polypeptide, such as apoptosis. Catalytic activity can be measured, for example, by measuring the amount of phosphorylated substrate. Other activities, such as apoptosis, can be assessed using methods known in the art.

[0212] "Inhibitors" of a marker are used to refer to inhibitory molecules identified using in vitro and in vivo assays of a silent corticotroph adenoma biomarker (e.g., STAT3, phosphorylated STAT3). An "inhibitor" is a compound that, for example, binds to, partially blocks, or completely blocks the activity of, reduces, prevents, delays activation, inactivates, desensitizes, or downregulates the activity or expression of a silent corticotroph adenoma biomarker.

[0213] Inhibitors also include genetically modified silent ACTH adenoma biomarkers, e.g., forms with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, RNAi and siRNA molecules, small organic molecules, etc. Such assays for inhibitors include, for example, expressing the silent ACTH adenoma biomarker in vitro, in cells or cell extracts, administering a putative modulator compound, and then determining the functional effect on activity, as described above.

[0214] Samples or assays containing a silent ACTH adenoma biomarker (e.g., STAT3, phosphorylated STAT3) treated with a potential inhibitor are compared to control samples without the inhibitor to determine the extent of altered activity. The control sample (untreated or treated with a known modulator) is assigned a relative protein activity value of 100%. Inhibition of a silent ACTH adenoma biomarker is achieved when the activity value relative to the control is approximately 80%, preferably 50%, and more preferably 25-0%.

[0215] Compound pharmaceutical composition and medicine kit

[0216] The present invention provides a compound pharmaceutical composition containing the active ingredients (a) an inhibitor of STAT3 or its protein phosphorylation; and (b) a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention can also be prepared into a powder for aerosol inhalation. A preferred dosage form is an injectable preparation. In addition, the pharmaceutical composition of the present invention can also be used in conjunction with other therapeutic agents.

[0217] The present invention also provides a drug kit for preventing and / or treating diseases related to abnormal skin structure, the drug kit comprising:

[0218] (a1) a first container, and an inhibitor of STAT3 or its protein phosphorylation, or a drug containing an inhibitor of STAT3 or its protein phosphorylation, located in the first container;

[0219] (b1) an optional second container, and a detection reagent for STAT3 or protein phosphorylation thereof located in the second container.

[0220] The pharmaceutical composition and kit of the present invention are suitable for preventing and / or treating silent ACTH adenoma.

[0221] The preparation of the present invention can be taken three times a day to once every ten days, or taken once every ten days in a sustained-release manner. The preferred mode is to take it once a day because it is convenient for the patient to adhere to it, thereby significantly improving the patient's compliance with the medication.

[0222] When taking the drug, the total daily dose generally used in most cases should be lower than (or equal to or slightly greater than) the usual daily dose of each single drug in a few cases. Of course, the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.

[0223] Treatment

[0224] The present invention also provides a method for preventing and / or treating silent adrenocorticotropic hormone adenoma using the above-mentioned active ingredients or corresponding drugs of the present invention, which comprises administering an effective amount of active ingredient (a) STAT3 or an inhibitor of its protein phosphorylation to a mammal, or administering a pharmaceutical composition containing the active ingredient (a).

[0225] When the active ingredient of the present invention is used for the above-mentioned purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in an isotonic medium). For example, these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient mixed with a carrier.

[0226] The active ingredient or pharmaceutical composition of the present invention can be administered by conventional routes, including but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration. Preferred routes of administration include oral administration, intramuscular administration or intravenous administration.

[0227] From the standpoint of ease of administration, preferred pharmaceutical compositions are liquid compositions, especially injections.

[0228] In addition, the active ingredients or drugs of the present invention can also be used in combination with other drugs for preventing and / or treating diseases related to skin structural abnormalities (such as glucocorticoids, minoxidil, retinoic acid preparations, antibiotics, growth factors, etc.).

[0229] Diagnostic methods

[0230] The present invention provides a method for predicting or diagnosing silent ACTH adenoma by detecting STAT3 expression or enrichment of STAT3 or its phosphorylation sites. The prediction or diagnosis comprises determining the level of expression or enrichment of a silent ACTH adenoma biomarker in a biological sample from an individual, and then comparing the level to a control baseline or range.

[0231] Typically, the baseline value represents the level of a polynucleotide or nucleic acid in a healthy individual who does not have or will not develop a silent corticotroph adenoma (determined by using biological samples such as other subtypes of pituitary tumors (including but not limited to prolactinomas, somatotroph tumors, thyrotroph tumors, silent PIT1 lineage tumors, gonadotroph tumors, silent SF1 lineage tumors and null cell tumors) and normal pituitary tissue).

[0232] An alteration in the expression or enrichment level of a silent ACTH adenoma biomarker can indicate that the patient is at risk for having a silent ACTH adenoma.Such methods can be used in conjunction with other diagnostic methods known in the art.

[0233] Inhibitors also include agents designed to reduce the level of mRNA encoding silent ACTH adenoma biomarker polypeptides (e.g., antisense molecules, ribozymes, DNA enzymes, small inhibitory RNAs, etc.) or agents that reduce the level of translation from mRNA (e.g., translation blockers, such as antisense molecules complementary to the translation start point or other sequences on the mRNA molecule). In some embodiments, known methods are used to identify nucleic acid sequences that inhibit silent ACTH adenoma biomarkers. Such inhibitors may include, but are not limited to, siRNA oligonucleotides and antisense oligonucleotides. Inhibitor siRNA or antisense oligonucleotides can be used to specifically target STAT3 expression.

[0234] In some embodiments, RNA interference is used to produce small double-stranded RNA, small interfering RNA (siRNA) or short hairpin RNA (shRNA) inhibitors to affect the expression of STAT3, which is generally carried out by cutting and destroying its homologous RNA. Small interfering RNA (siRNA or shRNA, both used interchangeably) is generally a double-stranded RNA of 19-22nt. siRNA can be obtained by chemical synthesis or by RNAi technology based on DNA vectors. Using siRNA technology based on DNA vectors, a small DNA insert (about 70bp, which encodes a short hairpin RNA targeting a target gene) is cloned into a commercially available vector. The vector comprising the insert can be transfected into cells and expresses the short hairpin RNA. Hairpin RNA is rapidly processed into a double-stranded RNA (siRNA) of 19-22nt by the cell mechanism. siRNA is generally inserted into a suitable RNAi vector because synthetically prepared siRNA tends to have lower stability and is not so effective in transfection.

[0235] siRNA can be prepared using the methods and algorithms described in, for example, the following references: Wang et al., (2004) A Web-based Design Center for Vector-based siRNA and siRNA cassette Bioinformatics. (In press); Khvorova et al., (2003) Cell. 115(2): 209-16; Harborth et al., (2003) Antisense Nucleic Acid Drug Dev. 13(2): 83-105; Reynolds et al., (2004) Nat Biotechnol. 22: 326-30; and Ui-Tei et al., (2004) Nucleic Acids Res. 32: 936-48. Other tools for constructing siRNA sequences are online tools, such as siRNA TargetFinder and ConstructBuilder, available from GenScript; Oligo Design and Analysis Tools, available from Integrated DNA Technologies; or siDESIGN™ Center, available from Dharmacon, Inc. It is recommended to use the ORF (open reading frame) as the target selection region, preferably 50-100 nt downstream of the start codon, to construct siRNA.

[0236] In the present invention, test compounds generally include small chemical molecules and peptides. Peptide inhibitors can be designed to simulate the natural ligand or binding partner of the targeted molecule. In the context of the present invention, exemplary peptide inhibitors of STAT3 can be designed from, for example, STAT3 dimerization partners.

[0237] The main advantages of the present invention include:

[0238] (1) The present invention discovered for the first time that the transcription factor "Signal Transducer And Activator Of Transcription 3" (STAT3) or its phosphorylation site is specifically enriched in silent ACTH adenomas. In addition, the present invention also unexpectedly found that STAT3 or its phosphorylation inhibitor can effectively inhibit silent ACTH adenomas.

[0239] (2) The present invention is the first to discover that by preparing targeted drugs targeting STAT3 or its protein phosphorylation sites, silent ACTH adenomas can be controlled or eliminated through drugs, thereby avoiding or reducing the need for patients with this subtype of tumor to undergo repeated surgery or radiotherapy after recurrence.

[0240] (3) The present invention provides the use of STAT3 or its phosphorylation inhibitor in the preparation of a drug for preventing or treating silent ACTH adenoma, as well as a pharmaceutical composition comprising STAT3 or its phosphorylation inhibitor, to provide new drug options and targets for the treatment of silent ACTH adenoma.

[0241] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0242] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0243] Example 1 Multi-omics sequencing of DNA, RNA, protein and protein modification levels was performed on 200 patients with pituitary adenomas, and it was found that STAT3 and its phosphorylation sites were enriched in silent ACTH adenomas.

[0244] 1.1 Whole-exome data collection

[0245] DNA was extracted from 5 mg of fresh tissue (tissues were obtained from Huashan Hospital, including all subtypes of pituitary tumors (including but not limited to prolactinoma, somatotrophoma, thyrotropinoma, silent PIT1 lineage tumors, gonadotrophoma, silent SF1 lineage tumors, null cell tumors, corticotroph tumors and silent corticotroph adenomas) and normal pituitary tissue) using a micro kit (Covaris LE220 Sonicator, purchased from Covaris). After quality inspection, 100 ng of genomic DNA was used to construct a library.

[0246] The library construction process mainly includes: fragmentation using a hydrodynamic shearing system to produce 180-280bp fragments, and the remaining overhangs are converted to blunt ends by nuclease / polymerase activity; after adenylation of the 3' ends of the DNA fragments, oligonucleotides are ligated; DNA fragments with adapter molecules attached to both ends are selectively enriched in a PCR reaction; after the PCR reaction, the library is hybridized with biotin-labeled probes in liquid phase, and then the exons of the gene are captured using magnetic beads with streptavidin; the captured library is enriched in a PCR reaction to add index tags in preparation for sequencing. The product is purified using the AMPure XP system and quantified using the Agilent High Sensitivity DNA Assay on the Agilent Bioanalyzer 2100 system.

[0247] The sequencing and data processing pipeline includes: sequencing the DNA library on the Illumina HiSeq platform and generating 150bp paired-end reads; downstream bioinformatics analysis is based on high-quality data, and QC statistics include total reads, sequencing depth, sequencing error rate, and percentage of Q30 reads; sequencing data are matched to the reference sequence, and valid sequencing data are mapped to the reference human genome (UCSC hg19) using Burrows-Wheeler Aligner software to obtain raw mapping results stored in BAM format; Samtools mpileup and bcftools are used for mutation filtering, including single nucleotide mutations and insertion mutations; and functional annotations are linked between gene mutations and diseases.

[0248] 1.2 Transcriptome Data Collection

[0249] Using a micro kit ( RNA was extracted from 5 mg of fresh tissue (from Huashan Hospital, all subtypes of pituitary tumors (including but not limited to prolactinoma, somatotrophoma, thyrotrophoma, silent PIT1 lineage tumors, gonadotrophoma, silent SF1 lineage tumors, null cell tumors, corticotroph tumors and silent adrenocorticotroph adenomas) and normal pituitary tissue) using RNA-Seq RT-PCR Reagent, purchased from Invitrogen. After quality inspection, 500 ng was used for subsequent library construction.

[0250] The library construction process includes: first removing ribosomal RNA, then fragmenting the RNA and combining it with random primers; synthesizing the first strand of cDNA, and then synthesizing the second strand of cDNA; after obtaining the chain-specific library, linker ligation is performed, and in order to ensure the consistency of binding during sequencing, a large fragment screening process is performed; PCR amplification, and finally the library is purified and the DNA library quality is tested.

[0251] Sequencing analysis is performed based on 12G sequencing data. The data processing process includes: the first step is to remove adapter contamination, generally using the software trim_galore; the second step is data alignment, using the official TCGA alignment software STAR to first build a genome index and then perform alignment; the third step is to use the bam file generated by the alignment to generate expression levels FPKM / TPM.

[0252] 1.3 Proteome and Modificationome Data Collection

[0253] Proteins were extracted from fresh samples (from Huashan Hospital, all subtypes of pituitary tumors (including but not limited to prolactinoma, somatotrophoma, thyrotropinoma, silent PIT1 lineage tumor, gonadotrophoma, silent SF1 lineage tumor, null cell tumor, adrenocorticotrophoma and silent adrenocorticotroph adenoma) and normal pituitary tissue) by reductive alkylation and FASP enzymatic digestion. Peptide samples were obtained after trypsin digestion. The peptides were ionized in the ion source and detected according to the different mass-to-charge ratios of the ions under the action of magnetic and electric fields. Data-dependent acquisition mode (DDA) was used. The peptides after protein digestion by protease were scanned by primary mass spectrometry, and then the peptide ions were selected for secondary fragmentation. After secondary mass spectrometry detection, the actual spectrum produced was matched with the theoretical spectrum in the database for scoring and strict card value, thereby inferring protein information. Before mass spectrometry identification, the phosphorylated proteome was analyzed using the Fe-NTA kit (High-Select TM Fe-NTA Phosphopeptide Enrichment Kit (purchased from Thermo Fisher Scientific) was used to enrich phosphorylated peptides, and then non-labeled quantitative large-scale proteomic identification was performed.

[0254] 1.4 Integrating genome, transcriptome, proteome, and phosphoproteomic data

[0255] (1) Perform quality control on the original files of genome, transcriptome, proteome, and phosphoproteomics obtained in step 1: FastQC (v0.11.9) was used for data quality control of genome and transcriptome, and Firmiana was used for quality control of protein and phosphorylation data.

[0256] (2) Use maftools software to analyze genomic data for mutations and copy number variations;

[0257] (3) Based on the consensus cluster analysis software, molecular typing of transcriptome, proteome, and phosphoproteome was constructed. The specific molecular typing parameters were as follows: reps = 1,000, pItem = 0.8, pFeature = 0.8, clusterAlg = "hc", distance = "spearman". Given the high consistency of molecular typing of transcriptome, proteome, and phosphoproteome, the protein typing with the greatest clinical translational value was selected for inclusion. 1716 proteins were screened and the 10 clinical subtypes were divided into 7 types: GH enriched, EMTPRO enriched, PRL enriched, TSH / silent PIT1 enriched, ACTH enriched, silent TPIT enriched, and SF1 / NULL enriched.

[0258] 1.5 Conclusion

[0259] By integrating multi-omics sequencing of genomics, transcriptomics, proteomics, and phosphoproteomics in 200 pituitary adenomas covering all clinicopathological subtypes, we found that the transcription factor Signal Transducer And Activator Of Transcription 3 (STAT3) and its phosphorylation sites were specifically enriched in silent ACTH adenomas ( Figure 2 ), suggesting that this site has the potential to be further explored and used as a therapeutic target for this subtype of tumor.

[0260] Example 2 In a cohort of 800 pituitary adenomas, STAT3 and its protein phosphorylation sites were enriched in silent ACTH adenomas by immunohistochemistry.

[0261] 2.1 Fabrication of tissue microarrays

[0262] 800 cases of numbered tumor tissues (from Huashan Hospital, all subtypes of pituitary tumors (including but not limited to prolactin cell tumors, somatotroph cell tumors, thyrotropin cell tumors, silent PIT1 lineage tumors, gonadotroph cell tumors, silent SF1 lineage tumors and null cell tumors) were fixed in 4% paraformaldehyde for 24 hours in more than 5 times the volume, dehydrated by gradient alcohol, transparentized by xylene and embedded in paraffin to obtain paraffin block specimens of single tumors. A representative area of ​​each tumor sample was obtained from the paraffin block using a sampling needle with a diameter of 2 mm, and filled into the paraffin mold of the tissue chip in sequence to obtain a tissue chip paraffin block. The tissue chip paraffin block was cut into 3 μM thickness using a paraffin slicer and transferred to an adhesive slide to obtain a tissue chip slide.

[0263] 2.2 Immunohistochemical staining

[0264] The tissue chip slides were placed in xylene for dewaxing and gradient alcohol for rehydration. Antigen retrieval solution (sodium citrate or trisEDTA) was used to perform heat-guided antigen retrieval on the rehydrated tissue chip slides. After washing with detergent, 0.3% hydrogen peroxide solution was used for peroxidase blocking, 5% fetal bovine serum was used for non-specific site blocking, and the primary antibody diluent (STAT3 antibody, purchased from Cell Signaling Technology, 9139; STAT3 phosphorylation antibody, purchased from abcam, ab32143) at a specified dilution ratio was used to incubate overnight at 4 degrees. Wash again, and horseradish peroxidase (HRP) secondary antibody (Dako REAL TM EnVision TM HRP rabbit / mouse) were incubated at room temperature for 20 min, washed and then developed using 3,3'-diaminobenzidine (DAB) colorimetric solution (DakoREAL TM DAB+CHROMOGEN and Dako REAL TM The cells were stained with K5007 substrate buffer (DAKO, Glostrup, Denmark) for 5 min. After counterstaining the cell nuclei with hematoxylin, the cells were dehydrated again with graded alcohol and xylene, and then mounted with glass slides and neutral gum.

[0265] 2.3 Quantitative analysis of immunohistochemistry

[0266] Prepared tissue sections were scanned full-size using a slide scanner (Ocus, Grundium, Tampere, Finland) and quantitatively analyzed using Qupath software. Specifically, a tissue microarray network was first established for each section, and the corresponding patient information was added to each point. The stain vector estimator was then used for preprocessing, a pixel classifier was used to identify tissue regions, and a cell detection tool was used to identify each cell in each tissue region. Finally, the immunohistochemical staining index (H score) for each tissue region was calculated based on the DAB positivity of each cell.

[0267] 2.4 Conclusion

[0268] By using STAT3 and STAT3 phosphorylation antibodies (STAT3 antibody, purchased from Cell Signaling Technology, 9139; STAT3 phosphorylation antibody, purchased from abcam, ab32143), we performed tissue microarray immunohistochemistry on a large cohort of pituitary adenomas of 800 patients. We confirmed the enrichment of STAT3 and its protein phosphorylation sites in silent ACTH adenomas at the protein level in a large sample ( Figure 3 ), confirming the widespread existence of this site and its feasibility as a therapeutic target.

[0269] Example 3: Through drug screening in primary tumor organoids and in vivo efficacy evaluation in tumor-bearing mouse models, it was determined that silent ACTH adenomas are sensitive to STAT3 and its phosphorylation inhibitors

[0270] 3.1 Construction of primary pituitary adenoma organoids

[0271] Fresh tumor tissues removed during surgery (from Huashan Hospital, all subtypes of pituitary tumors (including but not limited to prolactinomas, somatotrophomas, thyrotropinomas, silent PIT1 lineage tumors, gonadotrophomas, silent SF1 lineage tumors, null cell tumors, adrenocorticotroph tumors and silent adrenocorticotroph adenomas) were immediately immersed in tissue culture medium (Advanced DMEM / F12, purchased from Thermo), stored in a 4°C refrigerator or ice box, and quickly sent to the cell culture room for processing. Scissors were used to remove non-tumor tissue and necrotic tumors burned by electrocoagulation, and the remaining high-quality tumors were cut into 1 mm pieces. 2The tumor tissue was digested into multicellular clumps using tissue dissociation solution at 37 degrees Celsius, and was blown and observed several times during the process. After digestion to the appropriate degree, fetal bovine serum was used to terminate the digestion, and large interstitial blocks were filtered out through a 100μM cell strainer. After centrifugation to remove the supernatant, phosphate buffer was added and washed twice, and the tissue was resuspended on ice with an appropriate amount of matrigel, and equal amounts were inoculated into 96-well plates or 384-well plates. The well plate was placed in a 37-degree incubator for 30 minutes to allow the matrigel to solidify, and then an appropriate amount of culture medium was added.

[0272] 3.2 Drug Concentration Titration in Primary Tumor Organoids

[0273] STAT3 phosphorylation inhibitors (Napabucasin / BBI608, purchased from MedChemExpress, HY-13919; or STAT3-IN-1, purchased from MedChemExpress, HY-100753) were diluted in equal proportions (30 μM, 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, and 0.04 μM) and added to tumor organoids. The medium was changed every 3 days for 6 days. On the 6th day, all supernatants were aspirated, and 100 μl of Celltiter Glo 3D (Promega) diluted 1:1 with phosphate buffer was added to each well. The cells were incubated for 30 minutes, and the chemiluminescence function of the microplate reader was used to read the luminescence. Graphpad (Prism) software was used to analyze and plot drug sensitivity curves.

[0274] 3.3 In vivo efficacy evaluation in primary tumor organoid-bearing models

[0275] High-quality primary tumor organoids were collected and inoculated into the groin of immunodeficient NOD-SCID mice (purchased from Vital River) using a trocar puncture. The diameter of the transplanted tumor was measured using a vernier caliper. About one month after inoculation, the tumor volume reached 100-150 mm. 3 The animals were randomly divided into a control group (solvent), a STAT3 inhibitor group 1 (Napabucasin), and a STAT3 inhibitor group 2 (STAT3-IN-1), with 6 animals in each group. The drug was administered by gavage at a dose of 30 mg / kg / day, once every three days, with a volume of approximately 300 μl per dose. At the same time, the tumor diameter was measured every three days using a vernier caliper to dynamically observe the anti-tumor effect of the STAT3 inhibitor for 1 month. After the experiment, the tumor-bearing mice were immediately killed, the tumors were weighed, photographed, and frozen, and HE staining and immunohistochemistry experiments were performed.

[0276] 3.4 Conclusion

[0277] By culturing three primary silent ACTH-producing adenoma organoids and titrating drug concentrations, we found that STAT3 phosphorylation inhibitors could inhibit the activity of all tumor organoids to less than 1% at lower concentrations ( Figure 4 By evaluating the efficacy in vivo in tumor-bearing mice, we found that both STAT3 inhibitors could effectively inhibit tumor proliferation ( Figure 5 ).

[0278] The above results confirm that STAT3 phosphorylation inhibitors can be used as targeted drugs for silent ACTH adenomas.

[0279] In summary, we found that STAT3 and the phosphorylation of its protein sites are enriched in silent ACTH adenomas. The phosphorylation of STAT3 and its protein sites can be used as therapeutic targets for this subtype of tumor to prepare drugs, and open up drug treatment methods for this subtype of tumor in addition to surgery and radiotherapy.

[0280] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of an inhibitor of STAT3 or its protein phosphorylation, characterized in that: For the prevention and / or treatment of silent ACTH-producing adenomas.

2. The use according to claim 1, characterized in that The inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, gene editors, or a combination thereof.

3. The use according to claim 1, characterized in that The STAT3 protein phosphorylation includes phosphorylation of serine at position 726, and / or phosphorylation of serine at position 727, and / or phosphorylation of threonine at position 713, and / or phosphorylation of threonine at position 714, and / or phosphorylation of serine at position 691, and / or phosphorylation of tyrosine at position 704, and / or phosphorylation of tyrosine at position 705.

4. A pharmaceutical composition, characterized in that include: (a) inhibitors of STAT3 or its protein phosphorylation; and (b) a pharmaceutically acceptable carrier.

5. A medicine box, characterized in that: include: (a1) A first container, and an inhibitor of STAT3 or its protein phosphorylation, or a drug containing an inhibitor of STAT3 or its protein phosphorylation, located in the first container.

6. Use of the pharmaceutical composition according to claim 4 or the medicine kit according to claim 5, characterized in that: For the prevention and / or treatment of silent ACTH-producing adenomas.

7. A method for screening candidate substances for preventing and / or treating silent ACTH adenoma, characterized in that: The method comprises the steps of: (a) In a test group, a test compound is added to the cell culture system, and the expression level (E1) and / or activity (A1) of STAT3 protein in the cells of the test group is observed; and / or the enrichment level (F1) of STAT3 protein or STAT3 protein phosphorylation; in a control group, no test compound is added to the same cell culture system, and the expression level (E0) and / or activity (A0) of STAT3 protein in the cells of the control group is observed; and / or the enrichment level (F0) of STAT3 protein or STAT3 protein phosphorylation; Among them, if the expression level (E1) and / or activity (A1) of STAT3 protein in the cells of the test group; and / or the enrichment level (F1) of STAT3 protein or STAT3 protein phosphorylation is significantly lower than that of the control group, it indicates that the test compound is a candidate substance for preventing and / or treating silent adrenocorticotroph adenoma that has an inhibitory effect on the expression and / or activity of STAT3 or an inhibitory effect on the phosphorylation of STAT3 protein.

8. A method for screening candidate substances for preventing and / or treating silent ACTH adenoma, characterized in that: The method comprises the steps of: (i) contacting a candidate substance with a STAT3 protein; and (ii) determining the functional effect of the candidate substance on the STAT3 protein, wherein the functional effect indicates that the compound prevents and / or treats silent ACTH adenoma.

9. A use of STAT3 or its protein phosphorylation site, or a detection reagent thereof, characterized in that: (i) for use as a marker for detecting or diagnosing silent ACTH adenoma; and / or (ii) for use in preparing a diagnostic reagent or kit for detecting or diagnosing silent ACTH adenoma.

10. A diagnostic kit for detecting or diagnosing silent ACTH adenoma, characterized in that: The kit comprises a container containing a detection reagent for detecting STAT3 or its protein phosphorylation site; and a label or instructions indicating that the kit is used for detecting or diagnosing silent corticotroph adenoma.

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