Application of inhibiting THAP3 and SMYD3 in synergistic treatment of tumors

Abstract

The invention provides an application of inhibiting THAP3 and SMYD3 in synergistic treatment of tumors. In particular, the present invention provides a THAP3 inhibitor, and uses of an active ingredient combination comprising a THAP3 inhibitor and an SMYD3 inhibitor. According to the invention, it is found for the first time that a transcription factor THAP3 is highly related to mitochondrial electron transport chain gene expression, and THAP3 collects SMYD3 to an electron transport chain gene and up-regulates H3K4me3 in a region near a gene promoter so as to realize epigenetic regulation of target gene expression; by inhibiting THAP3 and / or SMYD3, mitochondrial biogenesis of tumor cells can be remarkably inhibited. Therefore, a new target is provided for treating and / or preventing tumors or cancers.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of inhibiting THAP3 and SMYD3 in synergistic treatment of tumors. Background Art

[0002] Mitochondria are the energy factories of eukaryotic cells. As highly specialized organelles, mitochondria are surrounded by a double membrane and contain an electron transport chain (ETC) to maintain oxidative phosphorylation and cellular energy production. Although mitochondria are thought to have originated from prokaryotes during evolution and retain organelle-specific genomes, most mitochondrial genes are encoded by the nuclear genome. Transcription factors play an important role in regulating gene expression. The human genome encodes approximately 1,600 transcription factors. Previous studies have identified some nuclear transcription factors or their co-regulators that control mitochondrial gene expression and biogenesis. For example, estrogen-related receptor α (ESRRA) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) were found to promote mitochondrial gene expression and biogenesis (Austin and St Pierre, 2012; Dings et al., 2022). Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) also increase the expression of mitochondrial respiration-related genes at the transcriptional level (Bennett et al., 2022). Therefore, it is very likely that there are more unknown transcription factors involved in the expression regulation of mitochondrial genes encoded by the nuclear genome.

[0003] The electron transport chain is a unique molecular machine located on the inner membrane of mitochondria. The ETC is responsible for pumping protons from the mitochondrial matrix to the intermembrane space, thereby establishing a proton gradient to generate energy. The electron transport chain includes five protein complexes: NADH:ubiquinone oxidoreductase (complex I, or CI), succinate dehydrogenase (CII), cytochrome bc1 oxidoreductase (CIII), cytochrome c oxidase (CIV), and ATP synthase (CV). Complex V helps protons flow back to the mitochondrial matrix and produce ATP. Therefore, the electron transport chain is essential for cellular respiration and energy production. Notably, the biogenesis of the electron transport chain occurs in two separate cellular compartments. Although the mitochondrial genome encodes only 13 electron transport chain components, most electron transport chain genes are located in the nuclear genome. At present, little is known about which transcription factors control the expression of electron transport chain genes.

[0004] Recent studies have found that transcription factors can interact with epigenetic modification enzymes to regulate target gene expression (Wu and Zhang, 2017). Specifically, transcription factors can recruit epigenetic modification enzymes and guide them to specific genes, enabling them to accurately modify the genomic DNA or histones of target genes (Rasmussen and Helin, 2016; Wang et al., 2015). Histone methylation is one of the important types of epigenetic modifications, which may activate or silence the expression of target genes depending on the modification site and method. In the past decade of research, histone methyltransferases or methylases have been systematically discovered and identified, but the role of histone methylases in mitochondrial respiration has not yet been fully clarified.

[0005] It is worth noting that the biogenesis of the electron transport chain is in a dysregulated state in many types of tumors (Liu and Shi, 2020; Raimondi et al., 2020; Zhao et al., 2023). The degree of dependence of different types of tumors on mitochondrial activity shows great heterogeneity. Mitochondrial biogenesis is significantly upregulated in some types of tumors (such as liver cancer, bile duct cancer, leukemia, breast cancer, lung cancer, etc.), so these tumors are relatively dependent on mitochondrial activity. It is expected that efficient tumor suppression effects can be achieved by intervening in mitochondrial biogenesis. Therefore, exploring the biogenesis of the mitochondrial electron transport chain plays an important role in discovering new targets for tumor treatment. Summary of the invention

[0006] The purpose of the present invention is to provide a new target for the treatment and / or prevention of tumors or cancers, and further to provide a new anti-tumor drug and a new method for the treatment and / or prevention of tumors or cancers.

[0007] In a first aspect of the present invention, there is provided a use of a THAP3 inhibitor for preparing a composition or a preparation for:

[0008] (1) inhibiting mitochondrial biogenesis; (2) downregulating mitochondrial electron transport chain gene expression; and / or (3) treating and / or preventing tumors or cancer.

[0009] In another preferred embodiment, the THAP3 inhibitor downregulates the expression of the THAP3 gene (including transcription level and protein level), and / or inhibits or blocks the functional activity of THAP3.

[0010] In another preferred embodiment, the THAP3 inhibitor is selected from the following group: small molecule compounds, polypeptides, nucleic acids (eg, shRNA, siRNA or sgRNA) or a combination thereof.

[0011] In another preferred embodiment, the THAP3 inhibitor is a shRNA specifically targeting the THAP3 gene, and the sequence of the shRNA is shown in SEQ ID NOs: 1 and 2, or as shown in SEQ ID NOs: 3 and 4.

[0012] In another preferred embodiment, the “down-regulating mitochondrial electron transport chain gene expression” includes reducing the histone methylation level in the region near the mitochondrial electron transport chain gene promoter, thereby down-regulating the electron transport chain gene expression at the epigenetic level.

[0013] In another preferred embodiment, the “down-regulating the expression of mitochondrial electron transport chain genes” includes reducing the H3K4me3 level in the region near the promoter of the mitochondrial electron transport chain genes.

[0014] In another preferred embodiment, the mitochondrial electron transport chain gene is selected from the following group: NDUFB10, SDHB, UQCRH, COX5B, ATP5ME, or a combination thereof.

[0015] In another preferred embodiment, the tumor or cancer has the following characteristics: the occurrence and / or progression of the tumor or cancer is particularly related to the abnormal regulation of mitochondrial biogenesis.

[0016] In another preferred embodiment, the tumor or cancer is selected from the following group: bile duct cancer, liver cancer, acute myeloid leukemia (AML), breast cancer, and lung cancer.

[0017] In another preferred embodiment, the composition or preparation further comprises a second active ingredient, and the second active ingredient is a SMYD3 inhibitor.

[0018] In another preferred embodiment, the SMYD3 inhibitor downregulates the expression of the SMYD3 gene (including transcription level and protein level), and / or inhibits or blocks the functional activity of SMYD3.

[0019] In another preferred embodiment, the SMYD3 inhibitor is selected from the following group: small molecule compounds, polypeptides, nucleic acids (eg, shRNA, siRNA or sgRNA) or a combination thereof.

[0020] In another preferred example, the SMYD3 inhibitor is a shRNA specifically targeting the SMYD3 gene, and the sequence of the shRNA is shown in SEQ ID NOs: 5 and 6, or as shown in SEQ ID NOs: 7 and 8.

[0021] In another preferred embodiment, the SMYD3 inhibitor is BCI-121.

[0022] The second aspect of the present invention provides a use of a combination of active ingredients for preparing a composition or a preparation, wherein the composition or the preparation is used for:

[0023] (1) inhibiting mitochondrial biogenesis; (2) downregulating mitochondrial electron transport chain gene expression; and / or (3) treating and / or preventing tumors or cancer;

[0024] The active ingredient combination comprises a first active ingredient, a THAP3 inhibitor, and a second active ingredient, a SMYD3 inhibitor.

[0025] In another preferred embodiment, the THAP3 inhibitor downregulates the expression of the THAP3 gene (including transcription level and protein level), and / or inhibits or blocks the functional activity of THAP3.

[0026] In another preferred embodiment, the THAP3 inhibitor is selected from the following group: small molecule compounds, polypeptides, nucleic acids (eg, shRNA, siRNA or sgRNA) or a combination thereof.

[0027] In another preferred embodiment, the SMYD3 inhibitor downregulates the expression of the SMYD3 gene (including transcription level and protein level), and / or inhibits or blocks the functional activity of SMYD3.

[0028] In another preferred embodiment, the SMYD3 inhibitor is selected from the following group: small molecule compounds, polypeptides, nucleic acids (eg, shRNA, siRNA or sgRNA) or a combination thereof.

[0029] In another preferred embodiment, the first active ingredient is shRNA specifically targeting the THAP3 gene, and the second active ingredient is shRNA specifically targeting the SMYD3 gene.

[0030] In another preferred embodiment, the first active ingredient is shRNA specifically targeting the THAP3 gene, and the second active ingredient is the SMYD3-specific chemical inhibitor BCI-121.

[0031] In another preferred example, the sequence of the shRNA specifically targeting the THAP3 gene is shown as SEQ ID NOs: 1 and 2, or as shown as SEQ ID NOs: 3 and 4.

[0032] In another preferred example, the sequence of the shRNA specifically targeting the SMYD3 gene is shown as SEQ ID NOs: 5 and 6, or as shown as SEQ ID NOs: 7 and 8.

[0033] The third aspect of the present invention provides an active ingredient combination, the active ingredient combination comprising:

[0034] (i) a first active ingredient, which is a THAP3 inhibitor; and

[0035] (ii) a second active ingredient, which is a SMYD3 inhibitor.

[0036] In another preferred embodiment, the THAP3 inhibitor and SMYD3 inhibitor are as described above.

[0037] In a fourth aspect, the present invention provides a pharmaceutical composition comprising: (a) a THAP3 inhibitor, (b) a SMYD3 inhibitor, and (c) a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the THAP3 inhibitor and SMYD3 inhibitor are as described above.

[0039] In another preferred embodiment, the pharmaceutical composition is used for: (1) inhibiting mitochondrial biogenesis; (2) downregulating mitochondrial electron transport chain gene expression; and / or (3) treating and / or preventing tumors or cancers.

[0040] In another preferred embodiment, the tumor or cancer has the following characteristics: the occurrence and / or progression of the tumor or cancer is particularly related to the abnormal regulation of mitochondrial biogenesis.

[0041] In another preferred embodiment, the tumor or cancer is selected from the following group: bile duct cancer, liver cancer, acute myeloid leukemia (AML), breast cancer, and lung cancer.

[0042] In another preferred embodiment, the pharmaceutical composition is prepared as a liquid preparation or a lyophilized preparation.

[0043] In another preferred embodiment, the pharmaceutical composition is prepared as an injection.

[0044] In another preferred embodiment, the administration routes of the pharmaceutical preparation include: intravenous injection, intravenous infusion, intratumoral injection, subcutaneous injection or intramuscular injection.

[0045] A fifth aspect of the present invention provides a medicine kit, comprising:

[0046] (C1) a first formulation in a first container, the first formulation comprising a THAP3 inhibitor as a first active ingredient, and a pharmaceutically acceptable carrier;

[0047] (C2) a second formulation in a second container, the second formulation comprising a SMYD3 inhibitor as a second active ingredient; and a pharmaceutically acceptable carrier; and

[0048] (C3) instructions, wherein the instructions indicate that the drug kit is used to treat and / or prevent tumors or cancers.

[0049] In another preferred embodiment, the first preparation and the second preparation are independent of each other.

[0050] In another preferred embodiment, the first preparation is a lyophilized preparation or a liquid preparation, preferably a liquid preparation.

[0051] In another preferred embodiment, the second preparation is a lyophilized preparation or a liquid preparation, preferably a liquid preparation.

[0052] In another preferred embodiment, the first preparation and the second preparation are both injections.

[0053] In another preferred example, the first preparation is administered before or after the second preparation, or is administered simultaneously with the second preparation.

[0054] The sixth aspect of the present invention provides a method for treating and / or preventing tumors or cancers, the method comprising the steps of administering a THAP3 inhibitor, or the active ingredient combination described in the third aspect of the present invention, or the pharmaceutical composition described in the fourth aspect of the present invention to a subject in need thereof.

[0055] In another preferred embodiment, the tumor or cancer has the following characteristics: the occurrence and / or progression of the tumor or cancer is particularly related to the abnormal regulation of mitochondrial biogenesis.

[0056] In another preferred embodiment, the tumor or cancer is selected from the following group: bile duct cancer, liver cancer, acute myeloid leukemia (AML), breast cancer, and lung cancer.

[0057] In another preferred embodiment, the subject in need thereof includes a human or a non-human mammal.

[0058] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 .THAP3 is a transcription factor that maintains mitochondrial respiration.

[0060] Among them, (A) The Spearman correlation coefficients of electron transport chain genes and human transcription factors were retrieved from cBioPortal, and the statistics are shown as the average of the correlation between each transcription factor and the ETC gene. (B) The top ten transcription factors that are positively correlated with electron transport chain genes in TCGA liver tumors are shown in the figure. (CE) Gene set enrichment analysis (GSEA) of THAP3 and oxidative phosphorylation pathway in different tumor datasets. (F) Control shRNA or two different shRNAs targeting THAP3 were stably expressed in Huh7 cells, and the knockdown efficiency of THAP3 was determined by western blotting. (G) Total RNA was extracted to determine mRNA expression in control cells or stable knockdown THAP3 cells, and the genes detected included representative genes of electron transport chain complex IV and mitochondrial autophagy pathway. (H) Fluorescence intensity of stable cell lines was quantified by staining with MitoTracker Green (MTG) and flow cytometry analysis. (I) Control cells and stable knockdown THAP3 cells were used for hippocampal metabolic flux detection to determine oxygen consumption rate (OCR). All data are shown as the mean ± SD of at least three independent experiments; *p < 0.05, **p < 0.01; ns indicates not significant.

[0061] Figure 2 .THAP3 interacts with SMYD3 to maintain mitochondrial respiration.

[0062] Among them, (A) The Spearman correlation coefficients of electron transport chain genes and human histone methylases were retrieved from cBioPortal. The average correlation coefficients of each methylase with the electron transport chain genes are shown. (B) HA-tagged methylases were co-expressed with Flag-tagged THAP3 in HEK293T cells. Protein interactions were determined by co-immunoprecipitation and western blotting. (C) The interaction between SMYD3 and THAP3 was determined in Huh7 cells by co-immunoprecipitation and western blotting experiments. THAP3 antibody was used for immunopurification and IgG was used as a negative control. (DF) Gene set enrichment analysis of SMYD3 and oxidative phosphorylation pathways in different tumor datasets. (G) shRNA targeting SMYD3 was stably expressed in Huh7 cells. The knockdown efficiency of SMYD3 was determined by western blotting experiments. (H) Total RNA was extracted to determine mRNA expression in control cells or cells with stable knockdown of SMYD3, and the expression levels of representative genes from the electron transport chain complex and mitophagy pathway were determined. (I) shRNA targeting SMYD3 or THAP3 was stably expressed in Huh7 cells, and knockdown efficiency was determined by Western blotting. (J) Total RNA was extracted to determine mRNA expression in control cells or stable knockdown cells. (K) Control cells and stable knockdown cells were stained with MitoTracker Green (MTG) and analyzed by flow cytometry, and the MTG fluorescence intensity was measured and normalized by cell number. (L) Control cells and stable knockdown cells were subjected to hippocampal metabolic flux assay to determine oxygen consumption rate (OCR). All data are shown as mean ± SD of at least three independent experiments; *p < 0.05, **p < 0.01; ns indicates not significant.

[0063] Figure 3 .THAP3 recruits SMYD3 to upregulate electron transport chain activity at the epigenetic level.

[0064] Among them, (AD) ChIP-qPCR assay was performed on Huh7 stable cell lines, SMYD3 antibody was used for chromatin immunoprecipitation, and IgG was used as a negative control. (EH) ChIP-qPCR assay was performed on Huh7 stable cell lines; THAP3 antibody was used for chromatin immunoprecipitation, and IgG was used as a negative control. (IL) ChIP-qPCR assay was performed on Huh7 stable cell lines; H3K4me3 antibody was used for chromatin immunoprecipitation, and IgG was used as a negative control. (M) Control cells and cells with stable knockdown of THAP3 were treated with SMYD3 inhibitor, and the protein expression of THAP3 and SMYD3 was determined by Western blotting. (NP) Cells with stable knockdown of THAP3 were treated with SMYD3 inhibitor and ChIP-qPCR analysis was performed; chromatin immunoprecipitation was performed using SMYD3, THAP3, and H3K4me3 antibodies. (Q) Total RNA was extracted from cells treated with SMYD3 inhibitor, and mRNA expression was determined. All data are shown as the mean ± SD of at least three independent experiments; *p < 0.05, **p < 0.01; ns indicates not significant.

[0065] Figure 4 .THAP3 and SMYD3 synergistically maintain tumor cell proliferation.

[0066] Among them, (A) Control cells or cells with stable knockdown of THAP3 were treated with SMYD3 inhibitors, and the growth curve of Huh7 was determined. (BC) Colony formation assay was performed on cells with stable knockdown of THAP3, and cells were treated with SMYD3 inhibitors to determine the relative colony number. The representative images of each group are shown. (DF) THAP3 and / or SMYD3 were stably knocked down in liver cancer cells Huh7, cholangiocarcinoma cells RBE, and leukemia cells MV411, and cell growth curves were determined. (GI) THAP3 and / or SMYD3 were stably knocked down in liver cancer cells Huh7, cholangiocarcinoma cells RBE, and leukemia cells MV411, and the colony or clone formation ability of cells was determined. All data are shown as the mean ± standard deviation of at least three independent experiments; *p<0.05, **p<0.01; ns indicates not significant. DETAILED DESCRIPTION

[0067] After extensive and in-depth research, the inventors unexpectedly discovered for the first time the correlation between the transcription factor THAP3 and the expression of mitochondrial electron transport chain genes. THAP3 is a transcription factor that maintains mitochondrial respiration, and further revealed the mechanism by which THAP3 regulates mitochondrial biogenesis. Specifically, the present invention analyzed the correlation between the electron transport chain and transcription factors in the TCGA tumor transcriptome data, and found that the transcription factor THAP3 was highly positively correlated with the electron transport chain. Mechanism studies have shown that THAP3 recruits SMYD3 to electron transport chain genes and upregulates H3K4me3 in the region near the gene promoter to epigenetically regulate target gene expression. The interaction between THAP3 and SMYD3 maintains the respiration, proliferation and cloning of tumor cells. In addition, inhibiting THAP3 and / or SMYD3 can significantly inhibit the mitochondrial biogenesis of tumor cells and inhibit the growth of tumor cells. Therefore, THAP3 and SMYD3 can be used as new targets for the treatment and / or prevention of tumors or cancers (especially tumors or cancers associated with abnormal regulation of mitochondrial biogenesis).

[0068] On this basis, the present invention has been completed.

[0069] the term

[0070] THAP3

[0071] THAP3, whose Chinese name is THAP domain protein 3 and whose English full name is THAP Domain Containing 3, is a transcription factor localized in the cell nucleus.

[0072] SMYD3

[0073] SMYD3, whose Chinese name is SET and MYND domain containing protein 3, and its English full name is SET And MYND Domain Containing 3, is a histone methyltransferase that can specifically catalyze the methylation of histone H3K4 and other sites, and can act as a transcription factor to regulate gene transcription expression and participate in signal transduction processes.

[0074] Mitochondrial biogenesis

[0075] Mitochondrial biogenesis refers to the process of mitochondrial formation and expansion, including mitochondrial genome replication, gene transcription and mitochondrial protein synthesis, as well as the biosynthesis of other mitochondrial components such as lipids and respiratory chain components.

[0076] As used herein, the term "abnormal regulation of mitochondrial biogenesis" includes: enhancement or reduction of mitochondrial biogenesis in tumor cells, epigenetic modification and transcriptional dysregulation of mitochondrial genes, abnormal regulation of mitochondrial protein expression and abnormal mitochondrial activity.

[0077] Active ingredient combination and pharmaceutical composition of the present invention

[0078] The present invention provides an active ingredient combination, which comprises a first active ingredient which is a THAP3 inhibitor, and a second active ingredient which is a SMYD3 inhibitor.

[0079] Based on the above combination, the present invention provides a pharmaceutical composition, which can be used for: (1) inhibiting mitochondrial biogenesis; (2) downregulating mitochondrial electron transport chain gene expression; and / or (3) treating and / or preventing tumors or cancer.

[0080] The pharmaceutical composition of the present invention comprises: (a) a THAP3 inhibitor, (b) a SMYD3 inhibitor, and (c) a pharmaceutically acceptable carrier.

[0081] Typically, the THAP3 inhibitor and / or SMYD3 inhibitor of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, wherein the pH is generally about 5-8, preferably, about pH 6-8.

[0082] As used herein, the term "THAP3 inhibitor" refers to an agent that can downregulate the expression of the THAP3 gene (including transcription level and protein level), and / or inhibit or block the functional activity of THAP3, including (but not limited to) small molecule compounds, polypeptides, nucleic acids (e.g., shRNA, siRNA or sgRNA). After the THAP3 inhibitor is administered to a subject, it downregulates the expression of mitochondrial electron transport chain genes by reducing the binding of THAP3 to target genes (mitochondrial electron transport chain genes) and reducing the recruitment of SMYD3 by THAP3, thereby inhibiting mitochondrial biogenesis. In a preferred embodiment of the present invention, the THAP3 inhibitor is an shRNA targeting THAP3.

[0083] As used herein, the term "SMYD3 inhibitor" refers to an agent that can downregulate the expression of the SMYD3 gene (including transcription level and protein level), or can completely or partially inhibit the activity of the SMYD3 methyltransferase, including (but not limited to) small molecule compounds, polypeptides, nucleic acids (e.g., shRNA, siRNA or sgRNA). After the SMYD3 inhibitor is administered to a subject, it downregulates the H3K4me3 level in the region near the promoter of the target gene, thereby downregulating the expression of mitochondrial electron transport chain genes and inhibiting mitochondrial biogenesis. In a preferred embodiment of the present invention, the SMYD3 inhibitor is an shRNA targeting SMYD3. In another preferred embodiment of the present invention, the SMYD3 inhibitor is a SMYD3-specific chemical inhibitor BCI-121.

[0084] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier used for administration of therapeutic agents, including various excipients and diluents.

[0085] The term "pharmaceutically acceptable carrier" refers to pharmaceutical carriers that are not essential active ingredients and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, and buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers. The carriers may also contain cell transfection reagents.

[0086] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and / or cells and can be accepted by humans and / or animals.

[0087] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration. The pharmaceutical preparation 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. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical combination of the present invention can also be prepared into a sustained-release preparation.

[0088] When using the pharmaceutical formulation, a safe and effective amount of the active ingredient combination (including THAP3 inhibitor and / or SMYD3 inhibitor) is administered to a mammal.

[0089] It should be understood that the effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; severity of the disease, weight of the patient, immune status of the patient, route of administration, etc.

[0090] There is no particular limitation on the administration method of the pharmaceutical composition of the present invention. Representative examples include (but are not limited to): intravenous injection, intravenous infusion, intratumoral injection, subcutaneous injection, intramuscular injection, etc.

[0091] Kit of the present invention

[0092] The present invention provides a medicine box, comprising:

[0093] (C1) a first formulation in a first container, the first formulation comprising a THAP3 inhibitor as a first active ingredient, and a pharmaceutically acceptable carrier;

[0094] (C2) a second formulation in a second container, the second formulation comprising a SMYD3 inhibitor as a second active ingredient; and a pharmaceutically acceptable carrier; and

[0095] (C3) instructions, the instructions indicating that the drug kit is used to treat and / or prevent tumors or cancers; and / or treat and / or prevent hyperinsulinemia. The first preparation includes (but is not limited to): lyophilized preparations, liquid preparations (such as injections).

[0096] The first preparation or the second preparation includes (but is not limited to): solid preparation (such as tablets, capsules, powders), liquid preparation.

[0097] Typically, the kit contains one or more (eg, at least two) unit dosage forms containing a THAP3 inhibitor and one or more (eg, at least two) unit dosage forms containing a SMYD3 inhibitor; preferably, 4-10 of each.

[0098] As used herein, the term "unit dosage form" means that for the convenience of administration, a composition is prepared into a dosage form required for a single administration, including but not limited to various solid doses (such as tablets), liquid doses, capsules, and sustained-release preparations.

[0099] The instructions provided by the present invention may include the following description: the method of using the drug kit is to use the unit dosage form containing the THAP3 inhibitor and the unit dosage form containing the SMYD3 inhibitor at the same time. In another preferred embodiment, the method of using the drug kit also includes using the unit dosage form containing the THAP3 inhibitor or the unit dosage form containing the SMYD3 inhibitor alone. The drug kit is particularly suitable for cancers related to abnormal regulation of mitochondrial biogenesis and highly dependent on mitochondrial respiration, such as cholangiocarcinoma, liver cancer, acute myeloid leukemia, etc.

[0100] The drug kit provided by the present invention is prepared by the following steps: placing a preparation containing a THAP3 inhibitor and a preparation containing a SMYD3 inhibitor, as well as instructions, together to form a drug kit. The preparation containing a THAP3 inhibitor preferably contains a unit dosage form of a THAP3 inhibitor, and the preparation containing a SMYD3 inhibitor preferably contains a unit dosage form of a SMYD3 inhibitor.

[0101] The step preferably comprises placing at least one unit dosage form containing a THAP3 inhibitor and at least one unit dosage form containing a SMYD3 inhibitor together with instructions to form a kit.

[0102] Wherein, the terms "THAP3 inhibitor" and "SMYD3 inhibitor" are as described above.

[0103] Uses of the present invention

[0104] The present invention provides a use of a THAP3 inhibitor for preparing a composition or preparation for treating and / or preventing tumors or cancers. The THAP3 inhibitor inhibits mitochondrial biogenesis by downregulating the expression of mitochondrial electron transport chain genes in tumor cells, thereby inhibiting the proliferation of tumor cells.

[0105] The present invention also provides a use of an active ingredient combination, which includes a THAP3 inhibitor and a SMYD3 inhibitor, for preparing a composition or preparation for treating and / or preventing tumors or cancer. The THAP3 inhibitor and the SMYD3 inhibitor respectively block the activities of THAP3 and SMYD3 in tumor cells, reduce the recruitment of SMYD3 to mitochondrial electron transport chain genes by THAP3, downregulate the H3K4me3 level of mitochondrial electron transport chain genes, thereby downregulating gene expression levels, inhibiting mitochondrial biogenesis, and thus jointly exerting the effect of inhibiting tumor cell proliferation.

[0106] The THAP3 inhibitor and active ingredient combination of the present invention are particularly suitable for a type of tumor or cancer: the occurrence and / or progression of the tumor or cancer is particularly related to the abnormal regulation of mitochondrial biogenesis. Therefore, the THAP3 inhibitor and active ingredient combination of the present invention are particularly suitable for treating and / or preventing bile duct cancer, liver cancer, acute myeloid leukemia (AML), breast cancer, lung cancer, etc.

[0107] The beneficial effects of the present invention include:

[0108] (1) The present invention discovered for the first time the correlation between the transcription factor THAP3 and the expression of mitochondrial electron transport chain genes, and verified through experiments that THAP3 is a transcription factor that maintains mitochondrial respiration, thereby proposing that THAP3 inhibitors can be used to inhibit mitochondrial biogenesis, thereby treating and / or preventing cancer or tumors (especially cancers or tumors that are highly dependent on mitochondrial respiration).

[0109] (2) The present invention further discovered the interaction between THAP3 and histone methyltransferase SMYD3, and confirmed that THAP3 recruits SMYD3 to upregulate the expression of electron transport chain genes at the epigenetic level. At the same time, experiments have shown that inhibiting THAP3 and SMYD3 can effectively inhibit the proliferation of various tumor cells. On this basis, the application of inhibiting THAP3 and SMYD3 in the synergistic treatment of cancer or tumors (especially cancer or tumors that are highly dependent on mitochondrial respiration) is proposed.

[0110] (3) The present invention reveals the important mechanism by which transcription factors regulate mitochondrial biogenesis through epigenetic regulation, providing a new target for the treatment and / or prevention of tumors or cancer.

[0111] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally in accordance with conventional regulations, such as the conditions described in J. Sambrook et al., Molecular Cloning Laboratory Guide (Fourth Edition) (Science Press Co., Ltd., 2017), or in accordance with the conditions described in the product manual provided by the product manufacturer. The materials, reagents, instruments, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0112] Materials and methods

[0113] 1. Plasmids and Reagents

[0114] cDNA encoding human full-length THAP3, SMYD3, PRMT1, SUV39H1, and KMT5C were cloned into Flag- or HA-tagged vectors (pLV-EF1a-IRES), and all expression vectors were verified by DNA sequencing. Plasmid transfection was performed by using Lipofectamine 3000 (ThermoFisher).

[0115] BCI-121 is a chemical inhibitor of SMYD3 (Selleckchem #S6833) and cells were treated at a concentration of 5 μM.

[0116] 2. Cell Culture

[0117] Huh7 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Biological Industries), penicillin, streptomycin, and 2 mM L-glutamine (Corning) at 37°C in a humidified environment with 5% CO2.

[0118] 3. Construction of Stable Cell Lines

[0119] pMKO.1-puro and pMKO.1-hygro vectors were used to construct shRNA targeting THAP3 and SMYD3. Retrovirus was packaged using a dual plasmid system (gag and vsvg). The cells were mixed with 8μg / mL polybrene and transduced with the virus. Stable cell lines were obtained after one week of selection in puromycin (4μg / mL) or hygromycin (200μg / mL). The knockdown efficiency was further determined by Western blotting. The antibody information was: THAP3 (Abclone, rabbit anti #A12899), SMYD3 (AbClon, rabbit anti #A2262) and β-actin (Abclone, mouse anti #AC004), all of which are commercial antibodies.

[0120] The shRNA sequence targeting THAP3 is as follows:

[0121]

[0122] The shRNA sequence targeting SMYD3 is as follows:

[0123]

[0124] 4. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0125] Total RNA was extracted from cultured cells using the EasyPure RNA Purification Kit (Transgen) and reverse transcribed using the QuantScript RT Kit (Tiangen). Real-time PCR was performed on cDNA using the SYBR Green qPCR Master Mix (EZBioscience) kit with gene-specific primers. Each PCR reaction was repeated in triplicate and β-actin was used as a control. T Methods The relative amount of cDNA was calculated.

[0126] 5. MitoTracker Green staining

[0127] To measure the amount of mitochondria, 5 x 10 cells were cultured with 50 nM MitoTracker Green (Invitrogen) at 37°C. 6 Each cell was stained for 30 minutes and analyzed by flow cytometry. Fluorescence intensity was quantified and mitochondrial mass was normalized to cell number.

[0128] 6. Chromatin Immunoprecipitation and Quantitative PCR (ChIP-qPCR)

[0129] Huh7 cells were cross-linked with 1% paraformaldehyde and sonicated. Antibodies against THAP3 (Abclone, A12899), SMYD3 (Abclone, A2262), histone H3K4me3 (Abclone, A22146), and control IgG (Abclone, AC005) were used to immunoprecipitate the sonicated chromatin. Protein A-agarose (Millipore) was used to enrich the antibody-chromatin complex, washed, and eluted. After reversing the cross-linking and treating with proteinase K, the immunoprecipitated DNA was extracted with phenol-chloroform and recovered by ethanol precipitation, and the DNA enrichment efficiency was quantified by qPCR.

[0130] 7. Cell Proliferation Assay

[0131] Huh7 cells were seeded in 10 cm culture dishes, with one million cells per dish in 10 ml of culture medium, and counted for 5 consecutive days. Live cells were counted after staining with trypan blue.

[0132] 8. Determination of clone forming ability

[0133] Huh7 cells were seeded in 6-well plates, with 1000 cells per well inoculated in 2 mL of culture medium. After 7-10 days, the cell colonies were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Colonies with more than 50 cells were counted and analyzed.

[0134] 9. Tumor genome correlation analysis

[0135] The Spearman coefficients of transcription factors and epigenetic enzymes with ETC genes were retrieved from cBioportal (www.cBioportal.org) (Gao et al., 2013).

[0136] 10. Statistical Analysis

[0137] Statistical analysis was performed using two-tailed unpaired Student's t test. All data shown are repeated results of at least three independent experiments (mean ± SD). p < 0.05 was considered statistically significant.

[0138] 11. qPCR primer sequences

[0139]

[0140]

[0141] Example 1. THAP3 is a transcription factor that maintains mitochondrial respiration

[0142] In order to analyze the relationship between transcription factors and mitochondrial respiratory activity, the inventors analyzed the correlation between the electron transport chain and transcription factor expression at the transcriptome level of various TCGA tumors. The human genome encodes at least 1639 transcription factors, of which 1596 transcription factors have clear Spearman correlation information with electron transport chain genes in the cBioportal database. It is noteworthy that many transcription factors are positively correlated with the expression of electron transport chain genes (Spearman correlation coefficient>0.3)( Figure 1 A). Mitochondria play an important role in maintaining liver tumor cell stemness and promoting tumor development (Chen et al., 2023; Lee et al., 2022; Li et al., 2022b). Notably, among all transcription factors, THAP3 ranked first in correlation with electron transport chain gene expression in TCGA hepatocellular carcinoma (LIHC) ( Figure 1 B).

[0143] To verify the relationship between THAP3 and mitochondrial respiration, gene set enrichment analysis (GSEA) was performed on the electron transport chain genes. In the Kyoto Encyclopedia of Genes and Genomes (KEGG), the electron transport chain is annotated as an oxidative phosphorylation pathway. The results showed that a positive correlation between THAP3 and oxidative phosphorylation was observed in TCGA liver cancer and two other independent tumor transcriptome studies (Li et al., 2022a; TCGA, 2017; Zhang et al., 2022)( Figure 1 CE). Therefore, it is speculated that THAP3 may be a positive regulator of electron transport chain biogenesis and mitochondrial respiration.

[0144] THAP3 was stably knocked down in the human hepatoma cell line Huh7 using two different shRNAs. Figure 1 F), we further measured the mRNA expression levels of genes from different electron transport chain complexes (CI-CV). The expression levels of these genes were significantly reduced in THAP3 knockdown cells ( Figure 1 G). In contrast, genes from the mitophagy pathway, including PINK1, PARK2, TBK1, and ATG5 (Liu et al., 2021; Lu et al., 2019), did not show significant changes after knockdown of THAP3 ( Figure 1 G).

[0145] In addition, the control cells and cells with stable THAP3 knockdown were stained with the fluorescent dye MitoTracker Green (MTG), and the mitochondrial fluorescence intensity was measured by flow cytometry. The results showed that the amount of mitochondria in cells was significantly reduced after THAP3 knockdown ( Figure 1H). More importantly, metabolic flux analysis showed that both the basal and maximal respiration rates of THAP3 knockdown cells were significantly reduced ( Figure 1 I).

[0146] Together, these data suggest that THAP3 maintains the expression of electron transport chain genes, mitochondrial mass, and cellular respiration rate.

[0147] Example 2. THAP3 interacts with SMYD3 to support mitochondrial respiration

[0148] Studies have shown that transcription factors can interact with epigenetic modification enzymes to regulate gene expression (Feinberg et al., 2016; Sindhu et al., 2012). Histone methylation is a widespread epigenetic modification that occurs on lysine or arginine residues. Modification of different residues in the histone tail can turn on or off the transcription of target genes. Therefore, the inventors focused on histone methylases for subsequent research.

[0149] Correlation analysis of TCGA transcriptomics revealed a positive correlation between the expression of electron transport chain genes and four different methylases, namely protein arginine methyltransferase 1 (PRMT1), SET and MYND domain-containing protein 3 (SMYD3), SUV39H1 histone lysine methyltransferase (SUV39H1), and lysine methyltransferase 5C (KMT5C) ( Figure 2 A). To determine whether THAP3 interacts with these methyltransferases to regulate histone methylation, expression vectors for Flag-tagged THAP3 and HA-tagged methyltransferases were constructed. THAP3 and four different methyltransferases were co-expressed in HEK293T cells and immunoprecipitated. Interestingly, THAP3 specifically interacted only with SMYD3, with no detectable binding to the other three methyltransferases ( Figure 2 B). Immunoprecipitation with THAP3 antibody revealed that THAP3 could be detected in Huh7 cells binding to SMYD3 ( Figure 2 C).

[0150] To verify the correlation between SMYD3 and the electron transport chain, GSEA analysis was performed in different tumor transcriptomics studies. Similar to THAP3, SMYD3 also showed a positive correlation with oxidative phosphorylation in different datasets (Li et al., 2022a; Yang et al., 2021b; Zhang et al., 2022)( Figure 2 DF). These results suggest that SMYD3 may interact with THAP3 to enhance cellular respiration.

[0151] To determine the role of SMYD3 in regulating the expression of electron transport chain genes, SMYD3 was stably knocked down by shRNA in Huh7 cells ( Figure 2 G). Knockdown of SMYD3 reduces the expression of multiple electron transport chain genes in complex IV ( Figure 2 H). As a control, multiple genes from the mitophagy pathway did not show significant changes ( Figure 2 H). These results further support a role for SMYD3 in maintaining electron transport chain gene expression.

[0152] Next, we further examined how the SMYD3-THAP3 interaction affects electron transport chain activity and cellular respiration. We stably expressed shRNA targeting THAP3 and SMYD3 in Huh7 cells ( Figure 2 I). Knockdown of THAP3 or SMYD3 significantly downregulated the mRNA expression of electron transport chain genes (NDUFB10, SDHB, COX5B, and ATP5ME) ( Figure 2 J). Importantly, MTG staining of Huh7 cells revealed that mitochondrial mass was significantly reduced in cells with THAP3 or SMYD3 knockdown ( Figure 2 K). Metabolic flux analysis further showed that the oxygen consumption rate of SMYD3 and THAP3 knockdown cells was significantly reduced ( Figure 2 L). Thus, THAP3 interacts with SMYD3 to maintain electron transport chain gene expression and mitochondrial respiration.

[0153] Example 3. THAP3 recruits SMYD3 to upregulate electron transport chain gene expression at the epigenetic level

[0154] SMYD3 is a histone H3K4 methylase that mediates H3K4 trimethylation (H3K4me3). H3K4me3 is a sign of activated gene expression. To understand how THAP3 and SMYD3 interact, chromatin immunoprecipitation (ChIP) was performed using antibodies against THAP3, SMYD3, and H3K4me3. Because the mRNA expression of NDUFB10, SDHB, UQCRH, and ATP5ME was significantly changed in THAP3 or SMYD3 knockdown cells, these genes were also included in the subsequent analysis.

[0155] ChIP results showed that the ChIP signal of SMYD3 antibody decreased to the background level after knocking down SMYD3, indicating that the antibody has specificity ( Figure 3 AD). Importantly, knockdown of THAP3 impaired the binding of SMYD3 to target genes ( Figure 3 AD). These data indicate that SMYD3 binding to electron transport chain target genes is dependent on THAP3.

[0156] We further tested the binding of THAP3 to electron transport chain genes in stable knockdown cells. Although knockdown of THAP3 reduced the ChIP efficiency of THAP3 antibody, knockdown of SMYD3 only slightly affected the binding of THAP3 to target genes (NDUFB10, SDHB, UQCRH, and ATP5ME). Figure 3 EH). These results suggest that THAP3 may recruit SMYD3 to epigenetically regulate electron transport chain genes. Of particular note, knockdown of SMYD3 or THAP3 reduced H3K4me3 levels of electron transport chain target genes ( Figure 3 These results suggest that SMYD3 and THAP3 epigenetically regulate the expression of electron transport chain genes in the same pathway.

[0157] To elucidate the role of SMYD3 methylase activity in regulating the expression of electron transport chain genes, cells with stable knockdown of THAP3 were treated with the SMYD3-specific chemical inhibitor BCI-121 (SMYD3i) (Wang et al., 2019). Inhibition of SMYD3 methylase activity did not affect the protein expression of THAP3 ( Figure 3 M). We further tested the binding status of SMYD3 and THAP3 using NDUFB10 as the detection target. The inhibitor of SMYD3 significantly weakened its binding with NDUFB10 ( Figure 3 N). At the same time, the effect of SMYD3 inhibitor on the binding of THAP3 to NDUFB10 gene was not obvious ( Figure 3 O). Importantly, the SMYD3 inhibitor significantly reduced the H3K4me3 level in the region near the NDUFB10 gene promoter ( Figure 3 P). In addition, SMYD3 inhibitor treatment of cells significantly downregulated the mRNA expressions of NDUFB10, SDHB, UQCRH, COX5B, and ATP5ME ( Figure 3 Q). In summary, transcription factor THAP3 can recruit SMYD3 to electron transport chain target genes and upregulate gene expression at the epigenetic level.

[0158] Example 4. THAP3 and SMYD3 synergistically maintain tumor cell proliferation

[0159] In this example, the biological function of THAP3-SMYD3 interaction was further investigated. Mitochondria maintain ATP production to meet the energy needs of tumor cells. Previous studies have shown that mitochondrial respiration is essential for maintaining tumor cell stemness (Wei et al., 2019). After stably expressing shRNA targeting THAP3 in Huh7 cells, the cells were treated with SMYD3 inhibitors and the proliferation rate of the cells was monitored. Knockdown of THAP3 or inhibition of SMYD3 slowed the growth of Huh7 and reduced the cell's clonogenic ability ( Figure 4 AC). Interestingly, knockdown of THAP3 and simultaneous treatment of cells with a SMYD3 inhibitor led to a further decrease in the proliferation rate of Huh7 cells ( Figure 4 AC). These results suggest that THAP3 and SMYD3 cooperate to maintain HCC cell growth and colony formation.

[0160] To further analyze the synergistic effect of THAP3 and SMYD3, shRNA targeting THAP3 and SMYD3 was stably expressed in liver cancer cells Huh7, cholangiocarcinoma cells RBE and leukemia cells MV411. Simultaneous knockdown of THAP3 and SMYD3 can significantly reduce the cell proliferation rate compared with knockdown of either gene alone ( Figure 4 DF) and colony forming ability ( Figure 4 GI).

[0161] The above results indicate that inhibition of THAP3 and SMYD3 can be used for synergistic treatment of various tumors.

[0162] discuss

[0163] Mitochondria play a fundamental role in cellular metabolism and energy production (Koch et al., 2021). Abnormal regulation of mitochondrial biogenesis is associated with a variety of human diseases, including tumors, neurodegenerative diseases, and cardiovascular diseases (Pfanner et al., 2019; Xu et al., 2021). So far, only a few nuclear regulatory factors have been identified to regulate mitochondrial biogenesis (Hao et al., 2021; Kropotov et al., 2007; Yin et al., 2020). The electron transport chain is essential for mitochondrial energy production. The present invention systematically explores the correlation between the electron transport chain and nuclear transcription factors and identifies THAP3 as a regulator of cellular respiration. It is worth noting that the current understanding of the biological function of THAP3 is not clear. The present invention found that THAP3 promotes the biogenesis of the electron transport chain. THAP3 recruits SMYD3 to target genes and upregulates H3K4me3 levels, thereby enhancing the function of the electron transport chain at the epigenetic level.

[0164] The expression of mitochondrial genes has been found to be controlled by epigenetic factors and cellular metabolism. For example, TEA domain transcription factor 4 (TEAD4) binds to and upregulates OXPHOS genes; arginine acts as a metabolic signal to upregulate histone acetylation, promoting the recruitment of TEAD4 and mitochondrial biogenesis (Chen et al., 2021). In addition, histone H3K18 deacetylase SIRT7 can negatively regulate mitochondrial gene expression (Mohrin et al., 2015). Glucose signals downregulate SIRT7 activity and upregulate histone H3K18 acetylation to promote mitochondrial biogenesis (Yan et al., 2018). The present invention found that THAP3 recruits SMYD3 to increase the H3K4me3 level of electron transport chain genes, and this process is likely to be regulated by cellular metabolic signals.

[0165] It is worth noting that previous studies have found that SMYD3 upregulates the expression of cell cycle-dependent kinase 2 (CDK2) and matrix metallopeptidase 2 (MMP2), accelerating the cell cycle and promoting tumor cell metastasis, respectively (Wang et al., 2019). In addition, SMYD3 can transcriptionally activate the expression of tumor-promoting genes to accelerate the occurrence and development of tumors (Sarris et al., 2016). In line with these findings, the present invention confirms that SMYD3 is involved in the remodeling process of mitochondrial biogenesis in tumor cells. Therefore, SMYD3 may have multiple downstream targets to promote tumor development.

[0166] Mitochondria are co-encoded by the nuclear and mitochondrial genomes. Electron transport chain components must be produced in proportion by both genomes to ensure the normal functioning of oxidative phosphorylation activity (Basu et al., 2020; Pierce et al., 2017). However, how nuclear transcription factors and mitochondria-specific transcription machinery are coordinated remains largely unknown. Although some nuclear factors have been reported to translocate into mitochondria (Chatterjee et al., 2016; Lee et al., 2005), whether nuclear transcription factors directly promote transcription in mitochondria remains to be further elucidated. Therefore, subsequent studies need to focus on whether THAP3 communicates with mitochondrial transcription factors to coordinately regulate the biogenesis of the electron transport chain.

[0167] Mitochondrial dysfunction is a key feature of tumors (Jin et al., 2020; Yang et al., 2021a). Because mitochondria play a key regulatory role in apoptosis, previous studies have focused on how oncogenic signals inhibit apoptotic pathways in tumors. Recent studies have focused on how tumor cells reshape mitochondrial metabolism. Although the Warburg effect emphasizes the importance of the glycolytic pathway in many tumors such as liver tumors, mitochondrial metabolism also plays a key role in promoting tumor development. For example, mitochondrial oxidative metabolism is essential for tumor stem cells and tumor metastasis (Raggi et al., 2021; Wang et al., 2020; Xie et al., 2022). Existing tumor genomics suggests that different types of tumors and different subtypes of the same tumor are specific in their dependence on mitochondrial respirosomes. Therefore, it is expected that new tumor therapeutic targets will be discovered by intervening in mitochondrial activity, especially intervening in mitochondrial biogenesis. It is worth noting that tumors such as liver cancer, cholangiocarcinoma, leukemia lung cancer and breast cancer show activation of mitochondrial biogenesis, suggesting that intervening in mitochondrial biogenesis is a potential strategy for treating these tumors. Inhibiting mitochondrial activity by intervening THAP3 showed a good anti-cancer effect in the above tumor-derived cell lines. Therefore, the results of the present invention suggest that tumor cells may mediate the remodeling of mitochondrial biogenesis through the THAP3-SMYD3-electron transport chain axis, which is also a key therapeutic target.

[0168] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a THAP3 inhibitor, characterized in that: For preparing a composition or preparation for: (1) inhibiting mitochondrial biogenesis; (2) downregulating mitochondrial electron transport chain gene expression; and / or (3) treating and / or preventing tumors or cancer.

2. The use according to claim 1, characterized in that The THAP3 inhibitor is selected from the group consisting of a small molecule compound, a polypeptide, a nucleic acid (eg, shRNA, siRNA or sgRNA) or a combination thereof.

3. The use according to claim 1, characterized in that The “down-regulating mitochondrial electron transport chain gene expression” includes reducing the histone methylation level in the region near the mitochondrial electron transport chain gene promoter, thereby down-regulating the electron transport chain gene expression at the epigenetic level; Preferably, the “downregulating mitochondrial electron transport chain gene expression” comprises reducing the H3K4me3 level in the region near the mitochondrial electron transport chain gene promoter.

4. The use according to claim 1, characterized in that The tumor or cancer has the following characteristics: the occurrence and / or progression of the tumor or cancer is particularly associated with abnormal regulation of mitochondrial biogenesis.

5. The use according to claim 4, characterized in that The tumor or cancer is selected from the group consisting of bile duct cancer, liver cancer, acute myeloid leukemia (AML), breast cancer, and lung cancer.

6. Use of an active ingredient combination, characterized in that: For preparing a composition or preparation for: (1) inhibiting mitochondrial biogenesis; (2) downregulating mitochondrial electron transport chain gene expression; and / or (3) treating and / or preventing tumors or cancer; The active ingredient combination comprises a first active ingredient, a THAP3 inhibitor, and a second active ingredient, a SMYD3 inhibitor.

7. The use according to claim 6, characterized in that The SMYD3 inhibitor is selected from the group consisting of a small molecule compound, a polypeptide, a nucleic acid (eg, shRNA, siRNA or sgRNA) or a combination thereof.

8. An active ingredient combination, characterized in that The active ingredient combination includes: (i) a first active ingredient, which is a THAP3 inhibitor; and (ii) a second active ingredient, wherein the second active ingredient is a SMYD3 inhibitor.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) a THAP3 inhibitor, (b) a SMYD3 inhibitor, and (c) a pharmaceutically acceptable carrier.

10. A medicine box, characterized in that: The medicine kit comprises: (C1) a first formulation in a first container, the first formulation comprising a THAP3 inhibitor as a first active ingredient, and a pharmaceutically acceptable carrier; (C2) a second formulation in a second container, the second formulation comprising a SMYD3 inhibitor as a second active ingredient; and a pharmaceutically acceptable carrier; and (C3) instructions, wherein the instructions indicate that the drug kit is used to treat and / or prevent tumors or cancers.

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