Use of FBP1 mRNA in tumor treatment

Abstract

A use of FBP1 mRNA in tumor treatment. Provided is a use of an FBP1 protein, an mRNA, or an expression vector thereof for preparing a preparation or a composition, the preparation or the composition being used for: (a) dephosphorylating TERT S227, and (b) treating tumors related to TERT S227 high phosphorylation. Also provided is a pharmaceutical composition containing the FBP1 protein, the mRNA, or the expression vector thereof. The pharmaceutical composition, used alone or in combination with other mRNAs or oncogene-silencing siRNAs, can effectively promote or synergistically promote tumor cell senescence, thereby enhancing the tumor treatment effect, and providing a new strategy for the prevention and / or treatment of cancers or tumors related to TERT S227 high phosphorylation and / or FBP1 low-expression.

Description

Application of FBP1 mRNA in tumor therapy Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to the application of FBP1 mRNA in tumor treatment. Background Art

[0002] Telomeres, composed of repetitive nucleotide sequences called TTAGGG, form the "cap" structure that maintains chromosome integrity. Telomeres gradually shorten during each round of DNA replication. Loss of telomere function can lead to chromosome end fusion during mitosis, forming bipartite chromosomes and subsequent anaphase bridges that cause chromosome breakage. The resulting two daughter cells each contain an asymmetric chromatid. The two chromatids without telomeres will trigger a cycle of breakage-fusion-bridges during the next mitosis, ultimately leading to aging and apoptosis in normal cells.

[0003] In cancer cells, telomere length and elongation primarily depend on highly activated telomerase, which synthesizes telomeric repeats in the majority of human cancers, as well as alternative telomere length regulation in 5%–15% of cancers. Telomerase activity is regulated by telomerase reverse transcriptase (TERT), the catalytic protein subunit of telomerase, which regulates telomerase activity at both the transcriptional and post-translational levels. Phosphorylation of TERT significantly increases telomerase activity. For catalytically active telomerase to be assembled, cytoplasmic TERT must be translocated to the nucleus. Akt-mediated phosphorylation of TERT at serine (Ser, S) 227 facilitates the binding of importin α to the TERT nuclear localization signal (NLS). However, it remains unclear whether TERT phosphorylation, and consequently, telomere function, is differentially regulated in normal and cancer cells.

[0004] Therefore, mediating cancer cell senescence and thus inducing cancer cell apoptosis is a new research direction in this field. However, it is still unclear in this field what effective pathways or methods can be used to mediate cell senescence and thus induce cancer cell apoptosis, thereby effectively treating and / or alleviating cancer.

[0005] Therefore, developing a new method that can effectively promote the senescence of cancer cells and inhibit cell proliferation, thereby effectively treating and / or alleviating cancer is of great significance to the field. Summary of the Invention

[0006] The present invention provides a new method that can effectively promote the senescence of cancer cells and effectively treat and / or alleviate cancer.

[0007] In a first aspect of the present invention, there is provided a use of an FBP1 protein, mRNA, or an expression vector thereof for preparing a preparation or composition for:

[0008] (a) Dephosphorylation of TERT S227;

[0009] (b) Treatment of tumors with TERT S227 hyperphosphorylation.

[0010] In another preferred embodiment, the TERT S227 hyperphosphorylation refers to that the detected TERT S227 phosphorylation level C1 is significantly higher than the normal value C0 of the TERT S227 phosphorylation level.

[0011] In another preferred embodiment, the significantly higher refers to: C1 / C0 ≥ 1.5, preferably ≥ 2, more preferably ≥ 3.

[0012] In another preferred embodiment, the tumor is a tumor cell with high phosphorylation of TERT S227 and / or low expression of FBP1.

[0013] In another preferred embodiment, the low expression of FBP1 refers to that the detected FBP1 expression level Y1 is significantly lower than the normal value Y0 of the FBP1 expression level.

[0014] In another preferred embodiment, the significantly lower ratio refers to: Y1 / Y0≤2 / 3, preferably ≤1 / 2, more preferably ≤1 / 3.

[0015] In another preferred embodiment, the tumor is selected from the following group: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0016] In another preferred embodiment, the preparation or composition is also used to treat tumors with low FBP1 expression.

[0017] In another preferred embodiment, the preparation or composition is used for:

[0018] (i) Reduced nuclear translocation of TERT;

[0019] (ii) reducing telomerase activity;

[0020] (iii) reducing the length of telomerase;

[0021] (iv) promoting tumor cell senescence;

[0022] (v) Inhibit the growth of tumor cells.

[0023] In another preferred embodiment, the preparation is a laboratory preparation.

[0024] In another preferred embodiment, the composition is a pharmaceutical composition.

[0025] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0026] (a1) FBP1 protein, mRNA, or its expression vector; and

[0027] (b) a pharmaceutically acceptable carrier.

[0028] In another preferred embodiment, the FBP1 protein, mRNA, or its expression vector is encapsulated in the pharmaceutically acceptable carrier.

[0029] In another preferred embodiment, the pharmaceutically acceptable carrier is lipid particles.

[0030] In another preferred embodiment, the pharmaceutically acceptable carrier is lipid nanoparticles (LNP).

[0031] In another preferred embodiment, the pharmaceutical composition is a lipid particle.

[0032] In another preferred embodiment, the pharmaceutical composition is lipid nanoparticles.

[0033] In another preferred embodiment, the lipid nanoparticles encapsulate the FBP1 protein, mRNA, or its expression vector.

[0034] In another preferred embodiment, the lipid nanoparticles further contain an element that targets tumor cells.

[0035] In another preferred embodiment, the FBP1 mRNA is selected from the group consisting of wild-type FBP1 mRNA, FBP1 mRNA variants, sequence-modified FBP1 mRNA, or a combination thereof.

[0036] In another preferred embodiment, the FBP1 mRNA nucleotide sequence comprises SEQ ID NO: 5

[0037] In another preferred embodiment, the pharmaceutical composition further comprises mRNA of other tumor suppressor genes.

[0038] In another preferred embodiment, the tumor suppressor gene is selected from the group consisting of PTEN, FBW7, p53, VHL, RB1, or a combination thereof.

[0039] In another preferred embodiment, the pharmaceutical composition further comprises mRNA selected from the group consisting of PTEN mRNA, FBW7 mRNA, p53 mRNA, VHL mRNA, RB1 mRNA, or a combination thereof.

[0040] In another preferred embodiment, the mRNA is an mRNA selected from the group consisting of wild-type mRNA, mRNA variants, sequence-modified mRNA, or a combination thereof.

[0041] In another preferred embodiment, the nucleotide sequence of the PTEN mRNA includes SEQ ID NO: 6.

[0042] In another preferred example, the nucleotide sequence of the FBW7 mRNA includes SEQ ID NO:7.

[0043] In another preferred embodiment, the nucleotide sequence of the p53 mRNA includes SEQ ID NO:8.

[0044] In another preferred example, the nucleotide sequence of the VHL mRNA includes SEQ ID NO:9.

[0045] In another preferred embodiment, the nucleotide sequence of the RB1 mRNA includes SEQ ID NO:10.

[0046] In another preferred embodiment, the pharmaceutical composition further comprises a nucleic acid drug that inhibits oncogenes.

[0047] In another preferred embodiment, the nucleic acid drug includes siRNA.

[0048] In another preferred embodiment, the oncogene comprises a gene mutation selected from the group consisting of KRAS G12C, KRAS G12D, p110αE542K, p110αE545K, p110αH1047R, BRAF V600E, EGFR L858R, EGFR L861Q, EGFR G719X, EGFR S768I, or a combination thereof.

[0049] In another preferred embodiment, the pharmaceutical composition further comprises an siRNA selected from the following group (including the siRNA sequence, its variants and the siRNA with modified sequence):

[0050] siKRAS G12C, siKRAS G12D, sip110αE542K, sip110αE545K, sip110αH1047R, siBRAF V600E, siEGFR L858R, siEGFR L861Q, siEGFR G719X, siEGFR S768I, or a combination thereof.

[0051] In another preferred embodiment, the siRNA includes: an siRNA sequence designed for a mutation, its variants, and the siRNA sequence after sequence modification.

[0052] In another preferred embodiment, the nucleotide sequence of the siKRAS G12C includes: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or a combination thereof.

[0053] In another preferred embodiment, the nucleotide sequence of the siKRAS G12D comprises: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or a combination thereof.

[0054] In another preferred example, the nucleotide sequence of sip110αE542K includes: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or a combination thereof.

[0055] In another preferred example, the nucleotide sequence of sip110αE545K includes: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a combination thereof.

[0056] In another preferred example, the nucleotide sequence of sip110αH1047R includes: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a combination thereof.

[0057] In another preferred embodiment, the nucleotide sequence of the siBRAF V600E comprises: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a combination thereof.

[0058] In another preferred embodiment, the nucleotide sequence of the siEGFR L858R includes: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or a combination thereof.

[0059] In another preferred embodiment, the nucleotide sequence of the siEGFR L861Q includes: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a combination thereof.

[0060] In another preferred embodiment, the nucleotide sequence of the siEGFR G719X includes: SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a combination thereof.

[0061] In another preferred embodiment, the nucleotide sequence of the siEGFR S768I includes: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or a combination thereof.

[0062] In another preferred embodiment, the pharmaceutical composition further contains additional drugs for treating tumors.

[0063] In another preferred embodiment, the lipid nanoparticles include: ionizable lipids, lecithin, phospholipids and PEG lipids.

[0064] In another preferred embodiment, the lipid nanoparticles are ionizable lipids.

[0065] In another preferred embodiment, the encapsulation efficiency is ≥80%, and the average particle size of the encapsulation solution after encapsulation is 50-150 nm.

[0066] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0067] In another preferred embodiment, the pharmaceutically acceptable excipient is selected from the following group: a diluent, an excipient, a surfactant, a lubricant, a disintegrant, or a combination thereof.

[0068] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection, lyophilized preparation, nebulized inhalation preparation, and smearable preparation.

[0069] In another preferred embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intratumoral, intramuscular, subcutaneous, or intraperitoneal injection.

[0070] In another preferred embodiment, the pharmaceutical composition is administered transdermally, such as by transdermal application or electrode introduction.

[0071] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating cancer or tumor.

[0072] In a third aspect of the present invention, a reagent combination is provided, comprising:

[0073] (a) FBP1 protein, mRNA, or its expression vector; and

[0074] (b) Detection reagent for detecting TERT S227 phosphorylation.

[0075] In another preferred embodiment, the detection reagent is a TERT S227 phosphorylated antibody or a TERT S227 non-phosphorylated antibody.

[0076] In another preferred embodiment, the detection reagent is a TERT S227 phosphorylation antibody.

[0077] In another preferred embodiment, the expression vector is a tumor-targeted expression vector.

[0078] In another preferred embodiment, the expression vector is a lipid nanoparticle (LNP) containing FBP1 mRNA.

[0079] In another preferred embodiment, the reagent combination is used to detect the phosphorylation level of TERT S227.

[0080] In a fourth aspect of the invention, there is provided a use of the pharmaceutical composition according to the second aspect of the invention for preparing a medicament for preventing and / or treating cancer or tumors.

[0081] In another preferred embodiment, the cancer or tumor is a cancer or tumor with high phosphorylation of TERT S227 and / or low expression of FBP1.

[0082] In another preferred embodiment, the cancer or tumor includes: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0083] In a fifth aspect of the present invention, a kit is provided, comprising the reagent combination as described in the third aspect of the present invention.

[0084] In another preferred embodiment, the kit further comprises a mutant FBP1 protein, mRNA or an expression vector thereof, and the mutant FBP1 protein, mRNA or an expression vector thereof has no dephosphorylation function.

[0085] In another preferred embodiment, the mutation of the mutant FBP1 protein, mRNA or its expression vector is selected from the following group: N273A, C129S, or a combination thereof.

[0086] In another preferred embodiment, the mutation site of the mutant FBP1 protein, mRNA or its expression vector is selected from the following group: N273, C129, R244, D128, or a combination thereof.

[0087] In a sixth aspect of the present invention, there is provided an in vitro dephosphorylation method comprising the steps of:

[0088] (s1) Phosphorylated TERT S227 protein is brought into contact with FBP1 protein, thereby dephosphorylating TERT S227.

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

[0090] In a seventh aspect of the present invention, a method for treating and / or alleviating tumors is provided, comprising the steps of:

[0091] (a1) administering a therapeutically effective amount of the pharmaceutical composition according to the second aspect of the present invention to a patient in need thereof.

[0092] In another preferred embodiment, the patient in need is a patient with high phosphorylation of TERT S227 and / or low expression of FBP1.

[0093] In another preferred embodiment, the patient in need suffers from a tumor selected from the following group: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, bone sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0094] 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

[0095] Figure 1a-j shows that FBP1 acts as a protein phosphatase to dephosphorylate TERT S227.

[0096] Figure 2a-f shows that FBP1 C129 mediates TERT dephosphorylation.

[0097] Figure 3a-e shows that FBP1 inhibits TERT nuclear translocation and activity.

[0098] Figure 4a-h shows that FBP1 inhibits tumor growth by suppressing TERT activity.

[0099] Figure 5a-c showed that FBP1 expression was negatively correlated with TERT S227 levels in ccRCC patients.

[0100] Figure 6a-d shows that FBP1 inhibits tumor growth and promotes mouse survival in liver cancer.

[0101] Figure 7a-k shows the safety evaluation of LNP in mice.

[0102] Figures 8a-c show that FBP1 inhibits tumor growth in renal cancer.

[0103] FIG9 shows that the combination of FBP1 mRNA and PTEN mRNA can enhance the tumor suppression effect.

[0104] FIG10 shows that the combined use of FBP1 mRNA and FBW7 mRNA can enhance the tumor suppression effect.

[0105] FIG11 shows that the combination of FBP1 mRNA and VHL mRNA can enhance the tumor inhibitory effect.

[0106] FIG12 shows that the combined use of FBP1 mRNA and p53 mRNA can enhance the tumor suppressive effect.

[0107] FIG13 shows that the combined use of FBP1 mRNA and RB1 mRNA can enhance the tumor suppressive effect.

[0108] FIG14 shows that the combination of FBP1 mRNA and siKRAS G12C can enhance tumor suppression.

[0109] FIG15 shows that the combination of FBP1 mRNA and siKRAS G12D can enhance tumor suppression.

[0110] FIG16 shows that the combination of FBP1 mRNA and sip110αE542K, sip110αE545K, and sip110αH1047R can enhance the tumor inhibitory effect.

[0111] FIG17 shows that the combination of FBP1 mRNA and siBRAF V600E can enhance tumor suppression.

[0112] FIG18 shows that the combination of FBP1 mRNA and siEGFR L858R can enhance the tumor inhibitory effect.

[0113] FIG19 shows that the combination of FBP1 mRNA and siEGFR L861Q, siEGFR G719X, or siEGFR S768I can enhance the tumor inhibitory effect. DETAILED DESCRIPTION

[0114] After extensive and in-depth research and extensive screening, the inventors unexpectedly discovered that TERT is regulated by the metabolic enzyme FBP1. FBP1 dephosphorylates TERT S227 (pS227), inhibiting TERT nuclear translocation and telomerase activity, thereby reducing telomere length, thereby inhibiting tumor cell proliferation and promoting tumor cell senescence. Specifically, the present invention's research has shown that LNP-mediated delivery of FBP1 mRNA significantly inhibits tumor growth in tumors such as liver cancer, kidney cancer, lung cancer, pancreatic cancer, breast cancer, melanoma, colorectal cancer, bile duct cancer, and gastric cancer. Furthermore, the present invention also demonstrates that targeting siRNAs (without targeting the corresponding wild-type protein) against cancer-promoting mutations present in patients also has a significant therapeutic effect. Furthermore, the combined delivery of FBP1 mRNA and the siRNA or other mRNAs with therapeutic or palliative effects exhibits a synergistic effect, further inhibiting tumor growth. This is the basis for the completion of the present invention.

[0115] FBP1

[0116] FBP1, commonly referred to as fructose-1,6-bisphosphatase 1, is an enzyme involved in gluconeogenesis, a metabolic pathway that generates glucose from non-carbohydrate precursors. FBP1 catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. This enzyme is crucial for maintaining blood glucose levels, particularly during fasting or when energy demands are high, requiring glucose synthesis from non-carbohydrate sources. Dysregulation of FBP1 activity is associated with various metabolic diseases, including diabetes and cancer.

[0117] In mammals, FBP1 and FBP2 encode the liver and muscle isoforms of FBPase, respectively. FBP1 is primarily expressed in the liver and kidneys, is universally deficient in almost 100% of clear cell renal carcinoma (ccRCC), and is downregulated in hepatocellular carcinoma (HCC) and other cancer types. Low FBP1 expression promotes liver cancer progression and is associated with poor overall survival and a higher rate of tumor recurrence.

[0118] Furthermore, studies have shown that FBP1 interacts with and inhibits hypoxia-inducible factors (HIFs) in ccRCC and enhancer of Zeste homolog 2 (EZH2) in HCC and ccRCC cells in an enzyme-independent manner. Furthermore, FBP1 acts as a protein phosphatase, dephosphorylating histone H3 to regulate gene expression. However, whether FBP1 enzymatic activity has other functions that directly regulate tumor cell proliferation remains unclear.

[0119] PTEN

[0120] PTEN (Phosphatase and tensin homolog) is an important tumor suppressor gene that encodes a phosphatase. Initially, PTEN was found to be a frequently mutated gene in many common human tumors. As research deepened, scientists discovered the importance of PTEN in the cell signaling network. As a phosphatase, it inhibits cell proliferation and promotes cell apoptosis by negatively regulating the PI3K / AKT signaling pathway. The PI3K / AKT pathway is crucial for processes such as cell proliferation, survival, and metabolism. In addition, PTEN has also been found to play a role in regulating the cell cycle, cell adhesion, and cell apoptosis. Research on PTEN not only plays a key role in understanding the occurrence and development of tumors, but also helps to discover new treatment strategies and drug targets, because abnormal PTEN function is closely related to the occurrence and development of various cancers.

[0121] FBW7

[0122] FBW7 (also known as F-box and WDrepeatdomain-containing 7) is an important protein that is a component of the ubiquitin ligase complex SCF (Skp1-Cullin-F-box protein). It plays a key regulatory role in cells, particularly in regulating protein degradation and cell cycle control. FBW7 interacts with other members of the SCF complex through its F-box domain to ubiquitinate target proteins, thereby marking them for degradation by the proteasome. This process plays a crucial regulatory role in the stability and function of various proteins in cells. In terms of cell cycle regulation, FBW7 has been found to regulate many cell cycle proteins, such as cyclin E, cyclin D, Myc, and Jun, controlling the progression of the cell cycle by regulating their degradation. In addition, FBW7 has been associated with mutations in many tumor-related genes, and is particularly associated with the occurrence and progression of some cancers. FBXW7 is mutated in 6% to 8% of tumors and is a good marker for determining prognosis. The mutation of FBXW7 is related to the mutation of the p53 gene. FBXW7 and p53 synergistically inhibit tumor invasion and metastasis, and it is also an important tumor suppressor gene.

[0123] VHL

[0124] The VHL gene refers to the Von Hippel-Lindau gene, an important gene in the human genome located on chromosome 3. The VHL gene encodes a protein called VHL protein, which performs an important regulatory role in cells. The main function of the VHL gene is to inhibit the growth and development of tumors. It plays an important role in processes such as angiogenesis and cell apoptosis by regulating the perception and stabilization of oxygen molecules in cells. When the VHL gene is mutated or missing, the regulation of these processes is lost, which may lead to the occurrence of tumors. Mutations in the VHL gene are one of the main causes of VHL disease (Von Hippel-Lindau disease). This hereditary disease can cause patients to develop a variety of tumors, including retinoblastoma, cerebrospinal cord tumors, hemophagocytoma, and cystic pancreatic lesions. Mutations in the VHL gene are also associated with other types of tumors, such as renal cell carcinoma.

[0125] Therefore, research on the VHL gene is of great significance. It not only helps to better understand the pathogenesis of VHL disease, but also provides important clues for the diagnosis, treatment, and prevention of related diseases. Genetic testing and genetic counseling can also help family members with VHL gene mutations to monitor and manage their condition and reduce the risk of related diseases.

[0126] p53

[0127] The p53 gene is a crucial tumor suppressor gene in the human genome. It is known as a "guardian gene" because its main function is to monitor the genetic stability of cells and, in response to DNA damage or other cellular stresses, prompt cells to enter a state of repair or apoptosis (cell self-destruction) to prevent abnormal cell proliferation.

[0128] The role of p53 gene in tumors is mainly manifested in the following aspects:

[0129] 1) Maintaining genomic stability: The p53 gene monitors DNA damage in cells, such as ultraviolet radiation and exposure to chemical carcinogens, and promotes DNA repair or guides cells into the apoptosis pathway, thereby preventing the accumulation of DNA damage and maintaining genomic stability.

[0130] 2) Inhibit abnormal cell proliferation: When cells suffer DNA damage or other abnormal conditions, the p53 gene can prevent the uncontrolled proliferation of abnormal cells by regulating cell cycle, cell proliferation and apoptosis, thereby preventing the occurrence of tumors.

[0131] 3) Promote apoptosis: When cells suffer from severe DNA damage or other severe cell stress, the p53 gene can promote cells to enter the apoptosis pathway to eliminate potential cancerous cells, thereby inhibiting tumor development.

[0132] In general, the p53 gene plays a very important role in cell biology and is one of the key factors in maintaining cell health and preventing cancer. Mutation or loss of function of the p53 gene is one of the important causes of tumorigenesis, and abnormalities of the p53 gene can be observed in many tumors.

[0133] Therefore, the p53 gene has become one of the key targets of many scientific studies and anticancer drug development due to its important role in cell biology and oncology.

[0134] RB1

[0135] The RB1 gene is a crucial gene in the human genome, located on chromosome 13. It encodes a protein called retinoblastoma protein (Rb protein). The discovery of the RB1 gene is closely linked to the pathogenesis of retinoblastoma, a childhood eye tumor. The primary function of the RB1 gene is to inhibit cell proliferation. It controls cell growth and division by regulating the progression of the cell cycle. Rb protein can interact with cell cycle proteins, particularly E2F transcription factors, preventing them from activating cell cycle-related genes, thereby inhibiting cells from entering the S phase (DNA synthesis).

[0136] The role of RB1 gene in cancer is mainly reflected in the following aspects:

[0137] 1) Tumor suppressor effect: The RB1 gene is considered a tumor suppressor gene. When the RB1 gene mutates or becomes inactivated, it loses its inhibitory effect on cell proliferation, allowing cells to proliferate uncontrollably, leading to the development of tumors.

[0138] 2) Retinoblastoma: Mutations in the RB1 gene are the primary cause of hereditary retinoblastoma. This childhood eye tumor is typically caused by a mutation in the RB1 gene that causes the bidirectional allele (i.e., the RB1 gene) to lose its normal tumor suppressor function, leading to excessive proliferation of retinoblastoma cells.

[0139] 3) Other cancers: In addition to retinoblastoma, abnormalities in the RB1 gene are also associated with other types of tumors, including osteosarcoma, breast cancer, prostate cancer, etc.

[0140] BRAF V600E mutation

[0141] The BRAF V600E mutation is a common oncogenic mutation that typically occurs in exon 15 of the BRAF gene. This mutation results in an amino acid change from glutamic acid (E) to glutamic acid (V) in the BRAF protein, hence the name V600E mutation. The protein kinase encoded by the BRAF gene plays an important role in cell signaling pathways, particularly the RAS / RAF / MEK / ERK pathway. This pathway is involved in processes such as cell proliferation, growth, and differentiation.

[0142] However, when the BRAF gene undergoes the V600E mutation, this pathway can become aberrantly activated, leading to excessive cell proliferation and the development of cancer. The BRAF V600E mutation has been widely observed in a variety of cancers, including melanoma, leukemia, thyroid cancer, colorectal cancer, and non-small cell lung cancer. The mechanism of this mutation is that the negative charge of the acidic glutamate residue imparts phospho-mimetic properties. This mimics the phosphorylation of the nearby threonine T599 and serine S602 residues within the BRAF activation domain, which activate the wild-type form of the protein. The mutant glutamate residue thus activates BRAF by inhibiting the interaction between the glycine-rich loop of BRAF and the activation domain. Reduced inhibition of BRAF leads to an increase in its basal activity, thus being oncogenic.

[0143] In clinical practice, the detection of BRAFV600E mutation can be used as one of the important indicators for diagnosing specific types of cancer, formulating individualized treatment plans, and evaluating treatment effects.

[0144] EGFR

[0145] The epidermal growth factor receptor (EGFR), also known as HER1 or ERBB1, is one of the four members of the ERBB receptor tyrosine kinase family. Due to its high expression and the enhanced downstream signaling pathways it activates, EGFR plays a crucial role in the proliferation, survival, angiogenesis, and migration of various cells, particularly epithelial cells. Mutations in the tyrosine kinase binding domain of the EGFR gene are considered a positive prognostic and predictive marker, and most patients with EGFR mutations respond well to tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib.

[0146] Recent clinical trials have also shown that for patients with EGFR mutation-positive non-small cell lung cancer, the use of TKIs instead of initial platinum-based chemotherapy has become the most effective first-line treatment. Exon 19 in-frame deletion mutations and exon 21 L858R mutations are the two most common EGFR sensitivity mutations, accounting for 90% of all clinically important mutations associated with EGFR sensitivity.

[0147] In addition, EGFR has other carcinogenic mutations including L861Q, G719X, S768I, etc.

[0148] KRAS

[0149] KRAS is a membrane-bound GTPase. When receiving extracellular signals, single-molecule KRAS GTPase performs downstream signal transduction by switching between the activated GTP-bound state and the inactivated GDP-bound state. Under normal conditions, when GTP is hydrolyzed, it is converted to the inactivated GDP-bound state. RAS-GTP in tumors continuously activates multiple downstream signaling pathways, including the RAS-RAF-MEK-ERK, PI3K-AKT-mTOR and RALGDS-RAL pathways. It plays an important role in regulating cell proliferation, differentiation and anti-apoptosis. 97% of KRAS mutations in NSCLC occur in exons 2 and 3, codons 12, 13, and 61, with the highest proportion being G12C (40%).

[0150] Multiple amino acid changes lead to heterogeneity in different KRAS mutant proteins and their biological behaviors. Different KRAS mutant subtypes affect different downstream pathways. G12C or KRAS G12V They often have Ral signaling pathway activation and are less dependent on AKT than wild-type KRAS. However, other subtypes of KRAS mutant proteins are more dependent on AKT for signal transduction than wild-type KRAS. G12D Activation of PI3K and MEK signaling pathways. KRAS G12C Lung cancer than KRAS G12D They have higher ERK1 / 2 phosphorylation levels. Based on whether they rely on the KRAS pathway to maintain tumor activity, they can be divided into two categories: one is KRAS-driven tumors, which are mostly well-differentiated epithelial phenotypes; the other is not dependent on KRAS to maintain tumor activity, which is common in epithelial-mesenchymal transition (EMT). KRAS G12C Mainly occurs in lung cancer, KRAS G12D It mainly occurs in colorectal cancer and pancreatic cancer.

[0151] PI3K

[0152] PI3K belongs to the lipid kinase family and is primarily divided into three classes (types I, II, and III). Type I phosphoinositide 3-kinases (Class I PI3Ks) are dimers consisting of a catalytic subunit and a regulatory subunit. Different catalytic subunits differentiate the PI3Ks into four subtypes: PI3Kα / β / γ / δ, corresponding to the catalytic subunits p110α / β / γ / δ (the corresponding genes for these subtypes are PIK3CA / B / G / D). Furthermore, Class I PI3Ks are further divided into Class IA (PI3Kα / β / δ) and Class IB (PI3Kγ), depending on the regulatory subunit and its function. The PIK3CA gene encodes the catalytic subunit p110α. Multiple mutations in p110α are found in cancer, primarily including E542K, E545K, and H1047R. These mutations lead to abnormal p110α function, thereby impairing the normal regulation of the PI3K signaling pathway.

[0153] siRNA of the present invention

[0154] Oncogenic mutations in genes or proteins such as BRAF, EGFR, KRAS, and PI3K increase the occurrence and development of malignant tumors, but their wild-type forms play an important role in normal cell signal transduction and biological functions. Therefore, the present invention envisions that only targeting oncogenic mutations in genes or proteins such as BRAF, EGFR, KRAS, and PI3K, rather than their wild-type forms, can achieve the purpose of treating diseases without affecting the maintenance of normal cell functions.

[0155] Therefore, in the present invention, the inventors designed siRNAs targeting only BRAF, EGFR, KRAS, and PI3K mutations, and delivered them to tumors through lipid nanoparticles to play a role in tumor treatment.

[0156] Pharmaceutical composition of the present invention and its application

[0157] In one aspect of the present invention, a pharmaceutical composition is also provided. In one embodiment of the present invention, the pharmaceutical composition is an mRNA pharmaceutical composition.

[0158] There are many methods for delivering mRNA. Scientists have established methods such as liposome delivery, polymer delivery, peptide chain delivery, virus-like replicon particle delivery, and cationic nanoemulsifier delivery. In addition, naked mRNA can also be injected directly into cells. The most commonly used delivery method is lipid nanoparticle delivery, which has the advantages of low toxicity and high delivery efficiency.

[0159] The "active ingredient" in the pharmaceutical composition of the present invention refers to the FBP1 mRNA described herein. In a preferred embodiment, the "active ingredient" further comprises an mRNA selected from the group consisting of PTEN mRNA, FBW7 mRNA, VHL mRNA, p53 mRNA, and RB1 mRNA. In a preferred embodiment, the "active ingredient" further comprises an siRNA selected from the group consisting of siKRAS G12C, siKRAS G12D, sip110αE542K, sip110αE545K, sip110αH1047R, siBRAF V600E, and siEGFR (L858R, L861Q, G719X, S768I).

[0160] The "active ingredients", preparations and / or compositions described in the present invention can be used to prevent and / or treat diseases or conditions such as infectious diseases, rare genetic diseases, neurodegenerative diseases, retinal diseases, cancers or tumors. "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

[0161] The pharmaceutical composition may be a liquid or a solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid.

[0162] Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0163] Pharmaceutical compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0164] The pharmaceutical composition of the present invention can be prepared into dosage forms such as injections, lyophilized agents, aerosol inhalers, and smear-type medicaments. The pharmaceutical composition of the present invention can be delivered (administered) in any suitable manner, including oral, parenteral, and topical methods. The pharmaceutical composition of the present invention can also be administered by injection, i.e., intravenous, intratumoral, intramuscular, intradermal, subcutaneous, or intraperitoneal injection. In addition, the pharmaceutical composition of the present invention can be administered transdermally. The transdermal administration method by the topical route can be formulated into applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, gels, paints, powders, and aerosols. In addition, the pharmaceutical composition of the present invention can be actively administered to positions such as intradermal, subcutaneous, muscle, tumor, tissue organs, and central nervous system through electrodes / electric fields / potential differences.

[0165] The pharmaceutical composition of the present invention can be co-administered with another activating agent. Co-administration is included in the administration of the compound of the present invention and activating agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours of each other. Co-administration also includes simultaneously, roughly simultaneously (for example, within about 1, 5, 10, 15, 20 or 30 minutes of each other) or sequentially administering the compound of the present invention and activating agent in any order. In some embodiments, co-administration can be completed by co-preparation, i.e., preparing a single pharmaceutical composition comprising the active ingredient of the present invention (mRNA transcription template construct of the present invention or optimized mRNA) and activating agent. In other embodiments, the active ingredient of the present invention and activating agent can be prepared separately.

[0166] The present invention also provides a use of the pharmaceutical composition for preparing a medicament for preventing and / or treating diseases, including (but not limited to) diseases characterized by hyperphosphorylation of TERT S227 and / or low expression of FBP1, such as cancers or tumors characterized by hyperphosphorylation of TERT S227 and / or low expression of FBP1. In a preferred embodiment, the cancer or tumor comprises:

[0167] The main advantages of the present invention include:

[0168] (1) The LNP-mediated FBP1 mRNA of the present invention can promote cell senescence and inhibit cell proliferation by mediating the dephosphorylation of TERT, thereby effectively mediating the apoptosis of tumor cells, and providing new strategies and ideas for exploring new cancer treatment methods based on the molecular typing of tumor patients in the clinic.

[0169] (2) The LNP-mediated FBP1 mRNA of the present invention can also be used in combination with other tumor suppressor gene mRNAs and siRNAs designed for cancer-promoting mutations to synergistically inhibit tumor growth, providing a new reference for clinical precision chemotherapy and targeted therapy.

[0170] 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 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.

[0171] Materials and methods

[0172] Cell culture

[0173] The cell lines Huh7, HEK-293T, HEK-293S, HK2, LN18, 786-O, A549, MIAPACA-2, H358, MCF7, MCF10A, T-47D, A375, and H3255 used in the present invention were obtained from authentic national cell culture collections and ATCC. Huh7, HK2, and HEK-293T cells were cultured in DMEM supplemented with 10% FBS (Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (Invitrogen, Thermo Fisher Scientific). 786-O cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. None of the cell lines used in the present invention were found in the Commonly Mistakenly Identified Cell Line Database maintained by the International Cell Line Authentication Committee or the BioSample database maintained by NCBI. These cell lines were authenticated by short tandem repeat typing and were found to be correct and free of mycoplasma contamination.

[0174] Protein immunoprecipitation

[0175] Cells were washed with cold PBS and centrifuged. Lysis buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 10% glycerol, 1 mM NaVO₃, 50 mM NaF, and a protease inhibitor cocktail) was added to the cell pellet and lysed at 4°C for 30 minutes. After centrifugation at 12,000 g, the supernatant was transferred to a pre-chilled centrifuge tube. 2 mg of protein was incubated with the corresponding antibody overnight and then incubated with protein A / G beads for 2 hours (or overnight with Flag-beads). The beads were washed three times with lysis buffer before immunoblotting analysis.

[0176] Protein purification

[0177] After constructing the prokaryotic expression vector, the E. coli BL21-DE3 strain was used to induce expression of the relevant protein. When the bacterial cells reached an OD value of 0.8-0.9 at 600 nm, IPTG was added to a final concentration of 0.1 mM. The bacterial growth temperature was lowered from 37°C to 16°C (for His-tag) or 25°C (for GST-tag). The cells were cultured overnight for an additional 16 hours, and then harvested by centrifugation. An appropriate amount of lysis buffer and nuclease was added to each tube, and the cells were incubated on ice for 30 minutes to degrade nucleic acids. The cells were disrupted by ultrasonication on ice. Centrifugation was performed at 12,000g for 30 minutes at 4°C. The supernatant was collected and used to bind to the corresponding beads. The proteins were then eluted using an imidazole gradient (for His-tag) or GSH (for GST-tag), concentrated, and the concentration was determined. Protein expression was then analyzed by SDS gel analysis, Coomassie blue staining, and western blot analysis. TERT protein was purified from HEK293S cells. Strep-MBP-TEV-tagged TERT was expressed in HEK293S cells. After centrifugation, the cultured HEK293S cells were supplemented with protease inhibitors (2 μg / mL DNase I, 0.5 μg / mL pepstatin, 2 μg / mL leupeptin, 1 μg / mL aprotinin, and 1 mM PMSF) and lysed by resuspending in Buffer 1 (20 mM Tris 8.0, 150 mM NaCl, 3 mM DTT). The cells were disrupted by sonication and incubated on ice for 60 minutes. The supernatant was then centrifuged at 10,000 g at 4°C, and the supernatant was incubated with streptavidin beads at 4°C for 90 minutes. The strep-MBP-TEV-TERT protein was eluted with 10 mM D-desthiobiotin. The eluted protein was cleaved overnight with recombinant TEV protease (v / v=1:20), and then the TERT protein was further separated and purified using AKTA (Superdex 200 column).

[0178] CRISPR / Cas9 system is a gene editing technology that knocks in mutations in tumor cells

[0179] CRISPR / Cas9 technology can be used to achieve site-directed gene knock-in mutagenesis, making experiments more efficient and accurate. The inventors will use this technology to perform knock-in mutagenesis in relevant cell lines. The specific experimental process includes gRNA design, CRISPR / Cas9 vector construction, donor vector construction, cell transfection, resistance screening, positive clone identification, monoclonal cell culture, and cell line construction.

[0180] TERT-pS227 antibody preparation

[0181] Rabbits were injected with the peptide RRGGS(phos)ASRS-C (SEQ ID NO:41) (KLH-conjugated) as an antigen. Five immunizations were performed. The first immunization dose was 0.75 mg, and subsequent immunizations used half the first dose. Finally, the serum was purified using the peptides RRGGS(phos)ASRS-C and RRGGSASRS-C.

[0182] Glutathione S-transferase pull-down assay (GST-pull down)

[0183] The corresponding proteins were mixed with glutathione magnetic beads in binding buffer (50 mM Tris-HCl, pH 7.5; 1% Triton X-100; 150 mM NaCl; 1 mM DTT; 0.5 mM EDTA; 100 μM PMSF; 100 μM leupeptin; 1 μM Maprotinin; 100 μM sodium oxyvanadate; 100 μM sodium pyrophosphate; 1 mM sodium fluoride) at 4°C overnight. The glutathione magnetic beads were washed three times with binding buffer and then analyzed by immunoblotting.

[0184] Metabolic flux analysis

[0185] Two million 786-O cells were plated per 10 cm dish and incubated with or without [U- 13 For mouse studies, [U- 13 C6-glucose was administered at a dose of 1.5 g / kg. Tissues were rapidly frozen in liquid nitrogen. Samples were thawed in an ice bath to minimize degradation. Each tube of cell sample was then added to 400 μl of 80% methanol and sonicated. The cells were then centrifuged at 14,000 g for 15 minutes at 4°C. The supernatant was collected, concentrated, and added to 100 μl of 80% methanol for LC-MS analysis.

[0186] Protein profiling

[0187] To identify interacting proteins, protein bands were cut from SDS-PAGE gels by Coomassie Brilliant Blue staining and digested with 200 ng of trypsin (Promega) at 37°C overnight in 50 mM ammonium hydroxide buffer containing RapiGest (Waters Corporation). The digested protein samples were analyzed using a highly sensitive LC-MS / MS with an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Protein identification was performed by comparing the fragment spectra with the UniProt protein database (EMBL-EBI) and using the Mascot search engine (version 2.3; Matrix Science) and the Proteome Discoverer software program (version 1.4; Thermo Fisher).

[0188] Immunofluorescence

[0189] Cells were fixed with 4% formaldehyde for 15 minutes and then treated with 0.25% Triton X-100 for 10 minutes to increase cell membrane permeability. After blocking with 5% bovine serum albumin (BSA) for 30 minutes, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with fluorochrome-conjugated secondary antibodies and DAPI. Immunofluorescence microscopy images were acquired using a Zeiss confocal microscope system (LSM900).

[0190] QFISH detection of telomere length

[0191] Cells were treated with 1 μg / mL colcemid for 90 minutes to maintain metaphase. Cells were incubated in cold 0.56% potassium chloride solution, fixed with methanol:acetic acid (3:1), plated on glass slides, and air-dried overnight. Slides were rehydrated with 2× SSC buffer and treated with 100 μg / mL RNase A for 1 hour, followed by treatment with pepsin (50 U / mL) for 10 minutes at 37°C. After 5 minutes of fixation in 4% formaldehyde, slides were dehydrated in 70%, 85%, and 100% ethanol for 1 minute each and air-dried. Slides were heated at 85°C for 5 minutes and hybridized with 200 nM TelC-Cy3 probe for 2 hours. After washing, coverslips were mounted on slides using ProLong Gold antifade reagent. Images were acquired using a Zeiss confocal microscope (LSM900) equipped with a 63× oil objective.

[0192] Telomere length determination by TRF analysis

[0193] Genomic DNA was isolated from the indicated cells using the QIAamp DNA mini kit (Qiagen). The isolated genomic DNA (2 mg) was digested with HinfI and RsaI (20 U each) overnight at 37°C. The DNA was separated by electrophoresis on a 0.8% agarose gel. The gel was denatured in 0.5 M NaOH and 1.5 M NaCl at 25°C with shaking for 30 minutes, then washed twice with 0.5 M Tris (pH 7.5) and 1.5 M NaCl at 25°C with shaking for 15 minutes each and neutralized. The gel was then transferred to a nylon membrane for southern blot. The gel was then crosslinked using a UV crosslinker at 120 mJ cm -2 DNA was crosslinked to the membrane using a 40°C setting. The membrane was prehybridized in DIG Easy Hyb Granules buffer for 1 hour, hybridized with a DIG-labeled telomere probe at 42°C for 4 hours, and then washed three times with 2× SSC buffer / 0.1% SDS, each for 15 minutes. The membrane was then incubated in a buffer containing DIG-AP antibody for 40 minutes and then developed using CSPD ready-to-use technology.

[0194] C-Circle Experiment

[0195] Genomic DNA was extracted using the QIAamp DNA mini kit (Qiagen). Diluted DNA (16 ng) was mixed with 0.2 mg / ml bovine serum albumin, 0.1% Tween, 4 mM dithiothreitol (DTT), 1 mM of each dNTP (except dCTP), 1×φ29 buffer (NEB), and 7.5 U φ29 DNA polymerase (NEB). The mixture was incubated at 30°C for 8 hours and then at 65°C for 20 minutes. Telomeric DNA levels were quantified using qPCR for samples treated or not treated with φ29 DNA polymerase (labeled as RCA+ and RCA-, respectively). C-Circle was calculated by ΔCt(RCA+) / ΔCt(RCA-).

[0196] The primers used in this assay are shown in Table 1 below.

[0197] Table 1

[0198] Phosphocysteine ​​intermediate capture experiment

[0199] Purified active AKT1 protein (SignalChem) (20 ng) was incubated with biotin-labeled TERT S227 peptide (1 mM) in 25 μl of kinase buffer (CST) and 10 μCi of [γ-32P]ATP at 30°C for 30 minutes. The reaction buffer was incubated with 20 μl of streptavidin magnetic beads to collect [γ-32P]ATP-labeled TERT S227 peptide, and the beads were then washed three times with phosphatase buffer (20 mM Tris–HCl (pH 8.5), 75 mM NaCl, 10 mM magnesium acetate, 0.57 mM EDTA, 0.033% BSA, and 1 mM DTT). WT His-FBP1 (1 μg) or His-FBP1 C129S (1 μg) protein was quickly mixed with [γ-32P]ATP-labeled TERT S227 peptide in 25 μl of phosphatase buffer at room temperature for 10 seconds. The phosphocysteine ​​intermediate capture experiment was terminated by adding SDS buffer and the samples were analyzed by SDS-PAGE without heating. 32 P-labeled covalently phosphocysteine ​​intermediates can be visualized by autoradiography.

[0200] Molecular dynamics simulation

[0201] The crystal structure of human liver FBP1 in complex with AMP (PDB: 5ZWK) was obtained from the Protein Data Bank (www.rcsb.org / ). The structure of TERT was predicted using AlphaFold (https: / / alphafold.ebi.ac.uk / entry / O14746). A set of one hundred models was generated, and the model with the lowest DOPE score was selected for further analysis. Subsequently, FBP1 was minimized and refined by molecular dynamics (MD) simulations using the Amber 16 software package and the ff14SB force field. The protein was solubilized in a truncated octahedral TIP3P water box containing 30,819 water molecules. Four Mg 2+ Ions are introduced to neutralize the system charge. The simulation begins with a two-stage minimization: First, the harmonic force that constrains the solute to its position is minimized by 3000 steps of steepest descent. Then 3000 steps of conjugate gradient 。 Then, in the NVT ensemble, the temperature was gradually increased from 50 to 300 K over 100 ps, ​​and the solute was Harmonic force potential constraints were applied. MD simulations were performed in the NPT ensemble, where positional constraints were gradually relaxed over 100 ps. Production runs lasted 1000 ns, with pressure coupling set to 1 atm and a constant temperature of 300 K. A 2 fs time step was used, and all hydrogen bonds were constrained using the SHAKE algorithm. Long-range electrostatic interactions were modeled using the particle mesh Ewald (PME) method. MD trajectories were analyzed using VMD (http: / / www.ks.uiuc.edu / ), and molecular representations were created using PyMOL (https: / / pymol.org / 2 / ).

[0202] Peptide-protein molecular docking

[0203] The crystal structure of human liver FBP1 in complex with AMP (PDB: 5ZWK) was retrieved from the Protein Data Bank. The structure of TERT was predicted using AlphaFold. The S227 phosphorylated TERT (TERT pS227) peptide was constructed and minimized using Schrodinger (https: / / www.schrodinger.com / ). Molecular docking of the FBP1 tetramer and the TERT pS227 peptide was performed using the HPEPDOCK web server (http: / / huanglab.phys.hust.edu.cn / hpepdock / ). The optimal binding mode was selected from the top 10 conformations obtained by docking for further analysis. The optimal binding mode was determined based on the docking score and experimental data. The docking results were analyzed using PyMOL.

[0204] Cell proliferation assay

[0205] Cell proliferation was assessed using the Cell Counting Kit-8 (CCK8). Cells were seeded at a density of 1,500 cells per well in a 96-well plate, and absorbance was measured at 450 nm.

[0206] Clone formation assay

[0207] Cells were seeded at a density of 4,000 cells per well in 6-well plates and cultured for 7-10 days. After washing with PBS, the cells were fixed with 4% formaldehyde for 15 minutes and then stained with 0.4% crystal violet for 20 minutes. After staining, the cells were washed with PBS and air-dried for visualization of colonies.

[0208] In vitro phosphatase assay

[0209] TERT pS227 polypeptide (Biotin-APGARRRGG-pS-ASRSLPLPKRP (SEQ ID NO: 42)) was synthesized by Nanjing Source Peptide Biotechnology Co., Ltd. Purified WT His-FBP1, His-FBP1C129S, His-FBP1 N273A, or His-FBP1G260R ​​protein (1 μg each) was incubated with TERT pS227 peptide (100 μM) in 25 μl of phosphatase reaction buffer (25 mM Tris-HCl, pH 7.4; 140 mM NaCl; 10 mM DTT) at 37°C for 30 minutes. Phosphate released from the polypeptide was measured using a malachite green assay kit (Sigma).

[0210] Telomerase assay

[0211] Telomerase activity was determined using the Telomerase Activity Quantification qPCR Assay Kit (ScienCell, #8928) according to the manufacturer's instructions.

[0212] FBP1 activity assay

[0213] FBP1 protein activity was measured using the BioVision Fructose-1,6-Bisphosphatase Activity Assay Kit (K590) according to the manufacturer's instructions. Km and Vmax values ​​were calculated using GraphPad Prism 10 software.

[0214] Cell senescence staining

[0215] β-Galactosidase staining was performed in 6-well plates (for in vitro studies) and on 12 μm frozen sections obtained from xenograft tumors using a β-galactosidase staining kit (Beyotime Biotechnology) according to the manufacturer's instructions.

[0216] Nuclear and cytoplasmic separation

[0217] Nuclear and cytoplasmic isolation was performed using a nuclear / cytoplasmic isolation kit (Beyotime Biotechnology) according to the manufacturer's instructions.

[0218] CRISPR / Cas9 system is a gene editing technology that knocks in mutations in tumor cells

[0219] CRISPR / Cas9 technology can be used to achieve site-directed gene knock-in mutagenesis, making experiments more efficient and accurate. The inventors will use this technology to perform knock-in mutagenesis in relevant cell lines. The specific experimental process includes gRNA design, CRISPR / Cas9 vector construction, donor vector construction, cell transfection, resistance screening, positive clone identification, monoclonal cell culture, and cell line construction.

[0220] LNP preparation

[0221] Ionized lipids SM102 and DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) were purchased from Shanghai Huabao Biochemical Co., Ltd.; DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-PEG2000 and DSPE-PEG2000-cRGD were purchased from Xi'an Ruixi Biotechnology Co., Ltd.; C16-TAT (C16-GGRKKRRQRRRPQ (SEQ ID NO: 43)) was purchased from Nanjing Yuanpeptide Biotechnology Co., Ltd.

[0222] Briefly, a lipid mixture (SM102, DSPC, cholesterol, and DSPE-PEG2000, at a molar ratio of 50:10:38.5:1.5) and a targeting peptide mixture (DSPE-PEG-cRGD, C16-TAT, and lipid mixture, at a weight ratio of 1:1:10) in ethanol were injected into a microfluidic device (Shanghai Weinano (Shanghai) Biotechnology Co., Ltd.) for self-assembly of LNPs. The formed LNPs were dialyzed twice against PBS buffer (pH 7.4) containing 2% sucrose for 12 hours each time to remove ethanol.

[0223] LNPs without FBP1 mRNA were used as control LNPs. The dose for animal studies was 0.5 mg / kg body weight.

[0224] Animal experiments

[0225] Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, approved by the Institutional Review Board of the First Affiliated Hospital, Zhejiang University School of Medicine. Six-week-old male nude mice were provided by Jicui Yaokang (Nanjing, China). Mice were housed under specific pathogen-free conditions with a 14-h light / 10-h dark cycle. The ambient temperature (21–23°C, 50% humidity) was maintained, with free access to water and feed. 2 × 10 6 Huh7 cells or 8×10 6 786-O cells were suspended in 75 μL DMEM or RPMI 1640 and injected subcutaneously with 75 μL matrix gel. Tumor size was measured every other day and the V (mm 3 )=0.52×length×width 2To calculate tumor volume.

[0226] Immunohistochemistry experiments

[0227] Human HCC (80 males and 10 females, aged 21-73 years) and ccRCC (58 males and 22 females, aged 35-75 years) samples, as well as adjacent normal tissue samples and clinical information were obtained from the First Affiliated Hospital, Zhejiang University School of Medicine.

[0228] The studies using human samples in this invention were approved by the Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine and complied with all relevant ethical regulations. All tissue samples were collected in accordance with the informed consent policy. All patients received standardized postoperative care. Paraffin sections of human samples were stained with antibodies against FBP1 or TERT pS227. Nonspecific IgG was used as a negative control. Tissue staining was quantitatively scored based on the percentage of positive cells and staining intensity.

[0229] Example 1 FBP1 dephosphorylates TERT S227

[0230] FBP1, a protein phosphatase, dephosphorylates TERT. To determine whether FBP1 possesses previously unknown tumor suppressor activity, this example immunoprecipitated FBP1 from the HCC cell line Huh7 cells and revealed an interaction between TERT and FBP1. This interaction was attenuated by knockdown of FBP1 expression using shRNA (Figure 1a). In vitro GST pulldown experiments demonstrated that purified bacterially expressed GST-FBP1 directly bound to Flag-TERT purified from 293T cells (Figure 1b). These results suggest that FBP1 directly interacts with TERT. Expression of various FBP1 truncation mutants in 786-O ccRCC cells revealed that the 236-275 truncation abolished FBP1 binding to TERT (Figure 1c). Mutations in this region to hydrophobic amino acids or surface-exposed residues revealed that IP experiments revealed that mutating aspartic acid (Asp, N) 273 to alanine (Ala, A) reduced FBP1 binding to TERT (Figure 1d). Furthermore, treatment of TERT immunoprecipitates with intestinal alkaline phosphatase (CIP) reduced the binding of TERT to FBP1 (Fig. 1e).

[0231] TERT activity is strictly controlled by phosphorylation. AKT-mediated phosphorylation of TERT serine (Ser, S) 227 transports TERT into the nucleus, and this nuclear transport is essential for the function of TERT. To determine whether FBP1 has a function different from metabolic activity and dephosphorylates TERT as a protein phosphatase, the present invention mixed purified active AKT1 with purified TERT expressed in HEK293S cells, added [γ-32P]ATP for protein phosphorylation experiments, and then incubated it with purified bacterially expressed His-FBP1 for protein dephosphorylation experiments. Radioautography showed that TERT phosphorylated by AKT1, but not the TERT S227A mutation, could be significantly dephosphorylated by FBP1 (Figure 1f).

[0232] Furthermore, immunoblotting analysis using a specific validated TERT pS227 antibody showed that FBP1 dephosphorylates S227-phosphorylated TERT but not TERT S227A (Figure 1g). Purified wild-type (WT) FBP1 and FBP1G260R ​​dephosphorylated TERT polypeptides containing S227 phosphorylation (Figure 1h). Correspondingly, overexpression of WT FBP1 and FBP1G260R, but not FBP1N273A, in 786-O ccRCC cells led to dephosphorylation of TERT at S227 (Figure 1i). In contrast, knockdown of FBP1 in Huh7 cells enhanced TERT S227 phosphorylation (Figure 1j).

[0233] Example 2 FBP1 C129 mediates TERT S227 dephosphorylation

[0234] The reduced cysteine ​​(Cys, C) in the catalytic domain of protein phosphatases is crucial for dephosphorylating protein substrates, and mutations in this cysteine ​​often create a "substrate trap," leading to increased binding of substrates to phosphatases. C129 of FBP1 is crucial for the dephosphorylation of histone H3. Molecular docking analysis of FBP1 and phosphorylated TERT peptide (Figure 2a) showed that TERT pS227 can be close to the catalytic domain of FBP1 ( ) docking at C129 (Figure 2a). Mutating C129 to serine (Ser) failed to dephosphorylate the TERT pS227 polypeptide (Figure 2b). Consistent with these in vitro results, expression of WT Flag-FBP1 inhibited the growth of 786-O cells (Figure 2c), indicating that FBP1 C129 is essential for dephosphorylation of TERT pS227.

[0235] Reduced cysteine ​​residues in the catalytic domain of protein phosphatases form a covalent phosphocysteine ​​intermediate, accompanied by rapid release of the dephosphorylated protein substrate. To determine the mechanism of FBP1-mediated TERT dephosphorylation, we conducted in vitro phosphorylation experiments. [γ-32P]-phosphorylated TERT S227 peptide was incubated with purified WT FBP1 or FBP1 C129S. The results showed that only WT FBP1 was labeled with [γ-32P] (Figure 2d). These results suggest that FBP1 forms a covalent phosphocysteine ​​intermediate during TERT pS227 dephosphorylation. To determine whether FBP1 requires the reduced C129S to dephosphorylate TERT pS227, we incubated purified FBP1 with purified AKT-phosphorylated TERT peptide in the presence of the oxidant H2O2. H2O2 treatment abolished FBP1-mediated dephosphorylation of TERT pS227 peptide (Figure 2e). However, the reducing agent dithiothreitol (DTT) abolished this effect.These results suggest that C129 of FBP1, in its reduced state, forms a covalent phosphocysteine ​​intermediate that plays a role in the dephosphorylation of TERT pS227.

[0236] Protein phosphatases with a catalytic cysteine ​​residue typically possess a CX5R (X can be any amino acid) motif, forming a pocket within the catalytic domain for substrate protein dephosphorylation. Although FBP1 lacks a CX5R, structural analysis revealed that C129, R244, and aspartic acid (Asp, D) 128 of FBP1 form a pocket, potentially interacting with the phosphate group of the phosphorylated TERT S227 peptide. Mutating R244 or D128 of FBP1 to alanine (Ala) reduced the ability of FBP1 to dephosphorylate the TERT S227 peptide (Figure 2f). These results provide ample evidence that FBP1 is a protein phosphatase that dephosphorylates TERT pS227.

[0237] Example 3 FBP1 inhibits TERT nuclear translocation and its function

[0238] Next, the present invention explored whether FBP1-mediated dephosphorylation of TERT pS227 affects the nuclear translocation of TERT. The present invention found that expression of WT FBP1, but not FBP1 C129S or FBP1 N273A, in 786-O cells reduced telomerase activity in a time-dependent manner (Figure 3a) and southern blot analysis (Figure 3b). Consistent with the effect of FBP1-regulated TERT dephosphorylation on telomere length in tumor cells, expression of WT FBP1 in 786-O cells increased cell senescence, as manifested by increased β-galactosidase activity (Figure 3c). These results indicate that FBP1 dephosphorylates TERT and inhibits its accumulation and activity in the cell nucleus, thereby shortening telomere length and inducing cell senescence.

[0239] Consistent with these results, expression of the phosphorylation-mimicking TERT S227D mutation in 786-O cells largely abolished WT FBP1-mediated inhibition of telomerase activity (Figure 3d) and reduction in telomere length (Figure 3e). These results suggest that FBP1 dephosphorylates TERT pS227, thereby reducing TERT nuclear accumulation, telomerase activity, and telomere length, and promoting tumor cell senescence.

[0240] Example 4 FBP1 inhibits tumor growth by inhibiting TERT activity

[0241] To determine the effect of FBP1 regulation of TERT on tumor cell proliferation, the present invention subcutaneously injected 786-O cells overexpressing WT FBP1, FBP1 C129S, or FBP1 N273A into nude mice. WT FBP1 expression significantly inhibited tumor growth (Figure 4a), accompanied by reduced tumor volume (Figure 4b) and tumor mass (Figure 4c), as well as enhanced cell senescence in the tumor (Figure 4d).

[0242] In contrast, FBP1 C129S and FBP1 N273A lost their effects in suppressing tumors and inducing cellular senescence (Figures 4a-d). Notably, in the presence of FBP1 deficiency and FBP1 C129S expression, re-expression of rTERT S227A in 786-O cells inhibited tumor growth to a lesser extent than in the presence of WT FBP1 expression (Figures 4e, 4f), suggesting that FBP1 has tumor suppressor functions that are both dependent on and independent of protein phosphatase activity.

[0243] In contrast, re-expression of rTERT S227D promoted tumor growth in the presence of FBP1 deficiency or expression of WT FBP1 and FBP1 C129S. These results suggest that FBP1 inhibits tumor growth by dephosphorylating TERT pS227.

[0244] Furthermore, hematoxylin-eosin (H&E) staining of tumor tissues revealed that overexpression of WT FBP1 increased anaphase bridge formation (Figure 4g), a hallmark of telomere dysfunction. Re-expression of TERT S227D, but not rTERT S227A, significantly attenuated the effect of WT FBP1 expression on anaphase bridge formation (Figure 4h). Thus, FBP1-mediated dephosphorylation of TERT disrupts telomere function, leading to tumor cell senescence and tumor growth inhibition.

[0245] Next, the present invention performed IHC analysis on 80 human ccRCC and adjacent normal tissues. Compared with normal tissues, decreased FBP1 and increased TERT S227 phosphorylation levels were detected in tumor specimens (Figure 5a). In addition, FBP1 expression levels were negatively correlated with TERT S227 phosphorylation levels in ccRCC and HCC specimens (Figure 5b). Notably, patients with higher levels of TERT S227 phosphorylation in their tumors had shorter survival than those with lower phosphorylation levels (Figure 5c).

[0246] These results support the clinical role of FBP1-regulated TERT dephosphorylation in human ccRCC and reveal the relationship between FBP1 expression, TERT S227 phosphorylation level and the clinical aggressiveness of ccRCC.

[0247] Example 5 FBP1 mRNA LNP delivery effectively inhibits tumor growth

[0248] The delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) as carriers has emerged as a novel therapeutic approach for the prevention and treatment of various diseases. To examine the therapeutic potential of FBP1, we designed ionized cationic lipids and encapsulated FBP1 mRNA (Figure 7a). Intravenous injection of LNPs into mice did not cause significant side effects, as reflected by H&E staining of heart, liver, spleen, lung, and kidney tissues (Figure 7b) and functional tests of the liver, heart, and kidneys (Figures 7c-h).

[0249] Will [U- 13 C6]-glucose injection into the tail vein of mice showed that LNP-FBP1 mRNA delivery significantly inhibited glycolytic flux in tumor tissue (Figure 7i) and moderately reduced glycolysis in the liver (Figure 7j), but not in the kidney (Figure 7k). As expected, LNP-WT FBP1 treatment significantly inhibited the growth of 786-O cell-derived tumors, accompanied by a decrease in tumor volume (Figure 8a) and weight (Figure 8b).

[0250] Furthermore, expression of WT FBP1, but not FBP1 C129S or FBP1 N273A, reduced TERT S227 phosphorylation levels (Figure 8c). WT FBP1-LNPs significantly reduced Ki67 expression compared to expression of FBP1 mutants (Figure 8c). Similarly, treatment with LNP-WT FBP1, but not LNP-FBP1 C129S or LNP-FBP1 N273A, significantly attenuated Huh7 cell-derived tumor growth (Figures 6a-b) and prolonged mouse survival (Figure 6c). Figure 6d shows the relationship between FBP1 and TERT S227 phosphorylation in normal and ccRCC cells, as well as in HCC cells, and the cellular processes they mediate.

[0251] These results indicate that LNP-mediated delivery of WT FBP1 mRNA effectively inhibited tumor growth and provided a promising therapeutic strategy for the treatment of ccRCC and HCC patients.

[0252] Example 6

[0253] In this example, the present invention investigates the therapeutic effects of lipid nanoparticle-mediated delivery of FBP1 mRNA, PTEN mRNA, and FBW7 mRNA alone and in combination in mouse animal models, as well as the therapeutic effects of lipid nanoparticle-mediated delivery of siRNA targeting KRAS G12C / G12D mutations, BRAF V600E mutations, EGFR L858R mutations, L861Q, G719X, S768I, p110αE542K, E545K, and H1047R mutations in mouse-related disease models.

[0254] 6.1 Experimental methods

[0255] A subcutaneous tumor model (cell-derived xenograft, CDX) was established in 6-week-old male nude mice using various cancer cell lines. 2x10 cells were injected. 6 Three days after the injection of FBP1 mRNA or FBP1 mRNA plus PTEN mRNA, FBW7 mRNA, VHL mRNA, p53 mRNA, and RB1 mRNA, lipid nanoparticles (LNPs) were injected into the tail vein. The mRNA was synthesized by in vitro transcription, capped and tailed, and the UTR was modified with pseudouridine and m5C. The injection was performed once every two days, and the tumor growth curve of the mice was observed and recorded.

[0256] siRNA sequences targeting KRAS G12C / G12D mutations, BRAF V600E mutations, EGFR L858R mutations, p110αE542K, E545K, and H1047R mutations were designed, and chemical modifications with methylation and cholesterol were performed to enhance their stability. Subcutaneous tumor models were established using breast cancer cell lines MCF7 (p110αE545K mutation), breast cancer cell line T-47D (p110αH1047R mutation), breast cancer cell line MCF10A (p110αE542K mutation) with gene knock-in mutation, lung cancer cell line H358 (KRAS G12C mutation), pancreatic cancer cell line MIAPACA-2 (KRAS G12D mutation), melanoma cell line A375 (BRAF V600E mutation), lung cancer cell line H3255 (EGFR L858R), and lung cancer cell line A549 with gene knock-in mutations (EGFR L861Q, G719X, S768I). 2x10 cells were injected. 6 Three days after the injection of FBP1 mRNA or FBP1 mRNA plus one of the above siRNAs, the mice were injected into the tail vein once every two days, and the tumor growth curve of the mice was observed and recorded.

[0257] The nucleic acid sequences used in this example are shown in Table 2 below.

[0258] Table 2

[0259] 6.2 Experimental Results

[0260] 6.2.1 LNP delivery of mRNA

[0261] A subcutaneous tumor-bearing model was established using the liver cancer cell line Huh7. As shown in Figure 9, LNP delivery of FBP1 mRNA or PTEN mRNA significantly inhibited tumor growth, and the combination of the two further inhibited tumor growth.

[0262] The results in Figure 10 show that LNP delivery of FBP1 mRNA or FBW7 mRNA significantly inhibited tumor growth, and the combination of the two could further inhibit tumor growth.

[0263] The results in Figure 11 show that LNP delivery of FBP1 mRNA or VHL mRNA significantly inhibited tumor growth, and the combination of the two could further inhibit tumor growth.

[0264] The results in Figure 12 show that LNP delivery of FBP1 mRNA or p53 mRNA significantly inhibited tumor growth, and the combination of the two could further inhibit tumor growth.

[0265] The results in Figure 13 show that LNP delivery of FBP1 mRNA or RB1 mRNA significantly inhibited tumor growth, and the combination of the two could further inhibit tumor growth.

[0266] In addition, the present invention also used pancreatic cancer cell lines, lung cancer cell lines, and breast cancer cell lines to construct subcutaneous tumor-bearing models and obtained similar results and consistent conclusions.

[0267] 6.2.2 LNP delivery of FBP1 mRNA and siRNA

[0268] A subcutaneous tumor-bearing mouse model was established using the lung cancer H358 cell line. As shown in Figure 14, LNP-delivered FBP1 mRNA or siKRAS G12C significantly inhibited tumor growth, and the combination of the two further suppressed tumor growth.

[0269] A subcutaneous tumor-bearing mouse model was established using the pancreatic cancer MIAPACA-2 cell line. As shown in Figure 15, LNP-delivered FBP1 mRNA or siKRAS G12D significantly inhibited tumor growth, and the combination of the two further suppressed tumor growth.

[0270] Subcutaneous tumor models were established in mice using the breast cancer cell lines MCF7 (p110αE545K mutation), T-47D (p110αH1047R mutation), and MCF10A (p110αE542K mutation), a knock-in breast cancer cell line. As shown in Figure 16, LNP delivery of FBP1 mRNA or single siP110αE542K, siP110αE545K, or siP110αH1047R significantly inhibited tumor growth. Combining FBP1 mRNA with siP110α further inhibited tumor growth.

[0271] A subcutaneous tumor model was established using melanoma A375 cells (BRAF V600E mutation). As shown in Figure 17, LNP delivery of FBP1 mRNA or siBRAF V600E significantly inhibited tumor growth, and the combination of the two further inhibited tumor growth.

[0272] A subcutaneous tumor-bearing model was established using the lung cancer cell line H3255 (EGFR L858R). As shown in Figure 18, LNP delivery of FBP1 mRNA or siEGFR L858R significantly inhibited tumor growth, and the combination of the two further inhibited tumor growth.

[0273] A subcutaneous tumor-bearing model was constructed using the lung cancer cell line A549 with gene knock-in mutations (EGFR L861Q, G719X, S768I). The results are shown in Figure 19. LNP delivery of FBP1 mRNA or siEGFR L861Q, G719X, S768I significantly inhibited tumor growth, and the combination of the two further inhibited tumor growth.

[0274] discuss

[0275] Telomere dysfunction promotes cancer development. Here, we demonstrate a previously unknown mechanism by which TERT is regulated by the metabolic enzyme FBP1. FBP1 directly interacts with TERT. Structural and mutational analyses identify the hydrophobic N273 of FBP1 as involved in binding to TERT. Importantly, FBP1 functions as a protein phosphatase and dephosphorylates TERT S227 in a manner that is dependent on FBP1 C129 but not metabolically active. Molecular dynamics simulations reveal that phosphorylated TERT S227 is closely adjacent to the FBP1 catalytic domain, forming a binding pocket with FBP1 C129 that binds to R244 and D128 of FBP1. FBP1 C129 in its reduced state generates a covalent phosphorylated-C129 intermediate, suggesting that transfer of the phosphate group from TERT pS227 to FBP C129, followed by release of dephosphorylated TERT, occurs before hydrolysis of the C129 thioether phosphate intermediate and after phosphate release. Dephosphorylated TERT fails to translocate to the nucleus, leading to inhibition of telomerase activity, reduced telomere length, suppressed tumor cell proliferation, and senescence, independent of ALT. FBP1 C129S or FBP1 N273A, although not altering FBP1 metabolic activity, largely failed to inhibit tumor growth, accompanied by increases in TERT S227 phosphorylation, telomere length, and Ki67 expression, as well as decreased anaphase bridge formation, which promotes tumor cell proliferation. The remaining tumor-suppressing effects of these mutants may be attributed to other functions of FBP1, either related or unrelated to its protein phosphatase activity, including its regulation of IκBα and AKT.

[0276] The human genome harbors at least 539 protein kinases that phosphorylate proteins, but only 189 identified protein phosphatases dephosphorylate them. This suggests that protein phosphatases are less specific in their substrate selection and that cells may utilize unknown and unrelated mechanisms to dephosphorylate proteins. Of the approximately 2,700 human enzymes, 1,653 are metabolic enzymes, far exceeding the total number of protein kinases and phosphatases. Numerous previous studies have demonstrated that metabolic enzymes can have additional functions and utilize proteins as substrates. It is well known that conventional protein phosphatases utilize reduced cysteines in their catalytic domains to form a covalent phosphorylation-cysteine ​​intermediate. This process is accompanied by the rapid release of the dephosphorylated protein substrate. Consistently, the present invention demonstrates that C129 of FBP1 is reduced and forms a covalent phosphorylation-C129 intermediate for dephosphorylation of TERT S227. Therefore, the additional activity of FBP1 shares a similar structural basis and catalytic mechanism with conventional protein phosphatases for dephosphorylating protein substrates. The present results are consistent with other publications, indicating that metabolic enzymes can have multifaceted roles and regulate protein substrates through direct dephosphorylation and phosphorylation.

[0277] Compared to normal cells, tumor cells utilize metabolic enzymes not only to serve cellular metabolic needs but also to direct numerous metabolically unrelated and instrumental cellular activities. Metabolic enzymes such as hexokinase 2 (HK2), phosphoenolpyruvate carboxylase 1 (PCK1), choline kinase α2 (CHKα2), 6-phosphofructokinase (PFKFB3), phosphoglycerate kinase 1 (PGK1), ketohexokinase (KHK) isoform A (KHK-A), and pyruvate kinase M2 (PKM2) have been shown to function as protein kinases to phosphorylate a variety of protein substrates, thereby promoting tumor growth. In the current report, we report that FBP1, originally described as a gluconeogenic enzyme, was found to be a bona fide protein phosphatase that dephosphorylates TERT by directly regulating cell division independently of FBP1's canonical metabolic function. Given that downregulation or absence of FBP1 expression frequently occurs in ccRCC, HCC, and many other cancer types and is often associated with advanced tumor stages, high-grade malignant phenotypes, and worse prognosis for cancer patients, the present findings provide key insights into the selective advantage of tumor cell immortalization due to the loss of FBP1-mediated telomerase inhibition. Thus, these findings reveal a previously unknown and critical mechanism by which FBP1 protein phosphatase activity differentially regulates TERT activity in tumor and normal cells. The present findings, which correlated decreased FBP1 expression with increased TERT S227 phosphorylation and shortened survival in ccRCC and HCC patients, as well as the potent tumor growth inhibition effect of LNP-based FBP1 mRNA delivery, highlight the potential for treating human cancers by modulating FBP1 protein phosphatase activity and expression.

[0278] The present study demonstrates that FBP1 interacts with TERT via its hydrophobic residue N273. FBP1 acts as a protein phosphatase and dephosphorylates TERT at S227 (pS227), thereby inhibiting TERT nuclear translocation and telomerase activity, reducing telomere length, and inhibiting tumor cell proliferation and promoting tumor cell senescence. Expression of a mutant deficient in FBP1 protein phosphatase activity promoted tumor growth in mice, accompanied by increases in TERT S227 phosphorylation and telomere length.

[0279] In addition, the present invention also demonstrates that LNP-mediated FBP1 mRNA delivery has a significant inhibitory effect on tumor growth in tumors such as liver cancer, kidney cancer, lung cancer, pancreatic cancer, breast cancer, and melanoma. At the same time, the present invention also has a good therapeutic effect by designing targeted siRNA (without targeting the corresponding wild-type protein) for cancer-promoting mutations present in patients. Combined with FBP1 mRNA delivery, it exhibits a synergistic effect and can better inhibit tumor growth. The results in these animal models provide new ideas and references for clinical precision therapy and targeted therapy.

[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. A use of FBP1 protein, mRNA, or its expression vector, characterized in that: For use in preparing a formulation or composition for: (a) Dephosphorylation of TERT S227; (b) Treatment of tumors with TERT S227 hyperphosphorylation.

2. The use according to claim 1, characterized in that The preparation or composition is used for: (i) Reduced nuclear translocation of TERT; (ii) reducing telomerase activity; (iii) reducing the length of telomerase; (iv) promoting tumor cell senescence; (v) Inhibit the growth of tumor cells.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a1) FBP1 protein, mRNA, or its expression vector; and (b) a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, wherein The pharmaceutical composition also includes: (a2) mRNA selected from the group consisting of PTEN mRNA, FBW7 mRNA, p53 mRNA, VHL mRNA, RB1 mRNA, or a combination thereof; and / or (a3) siRNA selected from the group consisting of siKRAS G12C, siKRAS G12D, sip110αE542K, sip110αE545K, sip110αH1047R, siBRAF V600E, siEGFR L858R, siEGFR L861Q, siEGFR G719X, siEGFR S768I, or a combination thereof; Wherein, the mRNA is selected from the following group: wild-type mRNA, mRNA variant, sequence-modified mRNA, or a combination thereof; and the siRNA is selected from the following group: siRNA sequence designed for mutation, its variant, sequence-modified siRNA sequence, or a combination thereof.

5. A reagent combination, characterized in that The reagent combination comprises: (a) FBP1 protein, mRNA, or its expression vector; and (b) Detection reagent for detecting TERT S227 phosphorylation.

6. The use of the pharmaceutical composition according to claim 3, characterized in that: Used for preparing medicines for preventing and / or treating cancer or tumors.

7. The use according to claim 6, characterized in that The cancer or tumor is a cancer or tumor with high phosphorylation of TERT S227 and / or low expression of FBP1.

8. The use according to claim 6, characterized in that The cancer or tumor includes: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

9. A kit, characterized in that The kit comprises the reagent combination according to claim 5.

10. An in vitro dephosphorylation method, characterized in that: Including steps: (s1) Phosphorylated TERT S227 protein is brought into contact with FBP1 protein, thereby dephosphorylating TERT S227.

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