Application of targeting SUCLG1 in treatment of mitochondrial generation dependent tumors

Abstract

The invention provides an application of a targeted succinyl-coenzyme A synthetase (SUCLG1) in treatment of mitochondrial generation dependent tumors. Specifically, the invention provides an application of an inhibitor targeting SUCLG1, and the inhibitor is used for preparing drugs for promoting succinylation modification of mitochondrial RNA polymerase (POLRMT) and inhibiting the activity of POLRMT so as to reduce mitochondrial biogenesis. And / or a composition or formulation for treating or preventing a mitochondrial biogenesis dependent tumor or cancer. The invention discloses an internal mechanism of the SUCLG1 for regulating mitochondrial biogenesis for the first time, and in-vivo and in-vitro experiments prove that the target inhibition of the SUCLG1 has a remarkable inhibition effect on the progress of mitochondrial generation dependent tumors, and a new target is provided for tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the application of targeting succinyl-CoA synthetase (SUCLG1) in treating mitochondriogenesis-dependent tumors. BACKGROUND

[0002] Mitochondria are double-membrane organelles and the energy center in eukaryotes. Unlike other organelles, mitochondria have a unique genome (mtDNA) that encodes essential components in the electron transport chain (ETC). mtDNA is subjected to mRNA synthesis and gene expression mediated by the organelle-specific transcription complex in the mitochondrial matrix. As a highly specialized organelle for energy production, mitochondria tightly couple mtDNA transcription with cellular metabolic status. However, the metabolic regulation mechanism of mtDNA remains unclear.

[0003] The mitochondrial transcription complex is composed of mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factors A and B2 (TFAM and TFB2M). TFAM and TFB2M facilitate the recognition of the transcription initiation site and the binding of POLRMT to mtDNA, which mediates the transcription of mtDNA-encoded genes. POLRMT is a key regulator of mtDNA replication and transcription, and it can also synthesize RNA primers required for mitochondrial genome replication. Studies have shown that the activity of the mitochondrial transcription complex is closely related to cellular metabolism: (1) mitochondrial gene expression is dynamically linked to nutritional supply and energy demand (Liesa & Shirihai, 2013; Scarpulla, 2008); (2) genetic mutations of various metabolic enzymes, such as succinyl-CoA synthetase subunit alpha (SUCLG1), succinyl-CoA synthetase [ADP-forming] beta subunit (SUCLA2), thymidine kinase 2 (TK2), and deoxyguanosine kinase (DGUOK), are clinically associated with mtDNA depletion syndrome (MDS) (Copeland, 2008). Notably, TK2 and DGUOK are regulators of nucleotide metabolism and provide precursors for mtDNA synthesis. In contrast, the regulatory role and mechanism of the tricarboxylic acid cycle enzymes SUCLG1 and SUCLA2 in mtDNA replication and transcription are not yet clear.

[0004] Mitochondria produce a variety of metabolic intermediates that can act as signaling molecules to regulate mitobiogenesis. Mitobiogenesis maintains energy production to support cell proliferation and tumor development. Oncogenic mutations reshape mitochondrial biogenesis, promoting tumorigenesis and progression. These intermediate metabolites can also serve as precursors for post-translational modifications (PTMs). Proteomic studies have shown that mtDNA replication and transcription complexes are modified in the form of phosphorylation, acetylation, and succinylation (King et al, 2018; Reardon & Mishanina, 2022; Wang et al., 2021). Notably, succinyl groups bind to lysine residues to form succinylated post-translational modifications, and succinyl-CoA provides succinyl groups to modify target proteins. Studies have shown that succinylation modification of metabolic enzymes regulates energy metabolism and redox balance in different tumors (Li et al, 2015; Tong et al, 2021; Zhou et al, 2016). However, the regulatory role of protein succinylation in mitobiogenesis is unclear. SUMMARY

[0005] The present application aims to provide the use of a SUCLG1-targeting inhibitor in the treatment of a mitochondria biogenesis-dependent tumor, and a method of treating a mitochondria biogenesis-dependent tumor with a SUCLG1-targeting inhibitor.

[0006] In a first aspect, the present application provides the use of a SUCLG1-targeting inhibitor for the preparation of a composition or a medicament for:

[0007] (1) promoting succinylation modification of mitochondrial RNA polymerase (POLRMT), inhibiting POLRMT activity, and thereby reducing mitochondrial biogenesis; and / or

[0008] (2) treating or preventing a mitochondria biogenesis-dependent tumor or cancer.

[0009] In another preferred embodiment, the SUCLG1-targeting inhibitor downregulates the expression (including the transcriptional level and the protein level) of the SUCLG1 gene, and / or inhibits or blocks the functional activity of the SUCLG1 protein.

[0010] In another preferred embodiment, the SUCLG1-targeting inhibitor is selected from the group consisting of a small molecule compound, a polypeptide, a nucleic acid (e.g., shRNA, siRNA, or sgRNA), or a combination thereof.

[0011] In another preferred embodiment, the inhibitor targeting SUCLG1 is an shRNA, the sequence of which is shown as SEQ ID NO: 1 and 2; or shown as SEQ ID NO: 3 and 4.

[0012] In another preferred embodiment, the inhibitor targeting SUCLG1 inhibits POLRMT activity by promoting the succinylation modification of the lysine site at position 622 (K622) of POLRMT.

[0013] In another preferred embodiment, the "inhibiting POLRMT activity" comprises: disrupting the binding of POLRMT to mitochondrial transcription factors TFAM and TFB2M; disrupting the binding of POLRMT to mtDNA; inhibiting the transcriptional expression of mitochondrial genes.

[0014] In another preferred embodiment, the mitochondrially-biogenesis-dependent tumor or cancer comprises (but is not limited to) acute myeloid leukemia, colorectal cancer, liver cancer, lung cancer, etc.

[0015] In another preferred embodiment, the mitochondrially-biogenesis-dependent tumor or cancer is acute myeloid leukemia, preferably FLT3-mutated acute myeloid leukemia.

[0016] In another preferred embodiment, the FLT3 mutation comprises tyrosine kinase domain (TKD) mutation and internal tandem duplication (ITD) mutation.

[0017] In another preferred embodiment, the composition is a pharmaceutical composition.

[0018] In another preferred embodiment, the pharmaceutical composition is prepared as a liquid formulation or a lyophilized formulation.

[0019] In another preferred embodiment, the pharmaceutical composition is prepared as an injection.

[0020] In another preferred embodiment, the administration route of the pharmaceutical composition comprises intravenous injection, intravenous infusion, intratumoral injection, subcutaneous injection or intramuscular injection.

[0021] In a second aspect of the present application, an active ingredient combination is provided, the active ingredient combination comprising:

[0022] (i) a first active ingredient, which is an inhibitor targeting SUCLG1.

[0023] In another preferred embodiment, the active ingredient combination further comprises (ii) a second active ingredient, which is an inhibitor targeting POLRMT.

[0024] In another preferred embodiment, the inhibitor targeting SUCLG1 down-regulates the expression (including transcriptional level and protein level) of SUCLG1 gene, and / or inhibits or blocks the functional activity of SUCLG1 protein.

[0025] In another preferred embodiment, the inhibitor targeting SUCLG1 is selected from the group consisting of a small molecule compound, a polypeptide, a nucleic acid (e.g., shRNA, siRNA or sgRNA), or a combination thereof.

[0026] In another preferred embodiment, the inhibitor targeting SUCLG1 is an shRNA, the sequence of which is shown in SEQ ID NO: 1 and 2; or shown in SEQ ID NO: 3 and 4.

[0027] In another preferred embodiment, the inhibitor targeting POLRMT down-regulates the expression (including transcriptional level and protein level) of POLRMT gene, and / or inhibits or blocks the functional activity of POLRMT protein.

[0028] In another preferred embodiment, the inhibitor targeting POLRMT is selected from the group consisting of a small molecule compound, a polypeptide, a nucleic acid (e.g., shRNA, siRNA or sgRNA), or a combination thereof.

[0029] In another preferred embodiment, the inhibitor targeting POLRMT is an shRNA, the sequence of which is shown in SEQ ID NO: 5 and 6.

[0030] In a third aspect, the present application provides use of an active ingredient combination for the preparation of a composition or formulation for: reducing mitochondrial biogenesis; and / or treating or preventing a mitochondrial biogenesis-dependent tumor or cancer.

[0031] In a third aspect, the present application provides use of an active ingredient combination for the preparation of a composition or formulation for: reducing mitochondrial biogenesis; and / or treating or preventing a mitochondrial biogenesis-dependent tumor or cancer.

[0032] In another preferred embodiment, the mitochondrial biogenesis-dependent tumor or cancer includes (but not limited to) acute myeloid leukemia, colorectal cancer, liver cancer, lung cancer, etc.

[0033] In another preferred embodiment, the mitochondrial biogenesis-dependent tumor or cancer is acute myeloid leukemia, preferably FLT3-mutated acute myeloid leukemia.

[0034] In another preferred embodiment, the FLT3 mutation includes tyrosine kinase domain (TKD) mutation and internal tandem duplication (ITD) mutation.

[0035] In another preferred embodiment, the inhibitor targeting SUCLG1 is as previously described.

[0036] In another preferred embodiment, the inhibitor targeting POLRMT is as previously described.

[0037] In a fourth aspect of the present application, there is provided a pharmaceutical composition comprising: (a) an inhibitor targeting SUCLG1, (b) an inhibitor targeting POLRMT, and (c) a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the pharmaceutical composition is used for: reducing mitochondrial biogenesis; and / or treating or preventing a mitochondrial biogenesis-dependent tumor or cancer.

[0039] In another preferred embodiment, the mitochondrial biogenesis-dependent tumor or cancer includes, but is not limited to, acute myeloid leukemia, colorectal cancer, liver cancer, lung cancer, etc.

[0040] In another preferred embodiment, the mitochondrial biogenesis-dependent tumor or cancer is acute myeloid leukemia, preferably FLT3-mutated acute myeloid leukemia.

[0041] In another preferred embodiment, the FLT3 mutation includes tyrosine kinase domain (TKD) mutation and internal tandem duplication (ITD) mutation.

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

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

[0044] In another preferred embodiment, the administration route of the pharmaceutical formulation includes intravenous injection, intravenous infusion, intratumoral injection, subcutaneous injection, or intramuscular injection.

[0045] In another preferred embodiment, the inhibitor targeting SUCLG1 is as previously described.

[0046] In another preferred embodiment, the inhibitor targeting POLRMT is as previously described.

[0047] In a fifth aspect of the present application, there is provided a kit comprising:

[0048] (C1) a first formulation contained in a first container, the first formulation comprising an inhibitor targeting SUCLG1 as a first active ingredient, and a pharmaceutically acceptable carrier;

[0049] (C2) a second formulation contained in a second container, the second formulation comprising an inhibitor targeting POLRMT as a second active ingredient; and a pharmaceutically acceptable carrier; and

[0050] (C3) a specification indicating that the kit is used for treating and / or preventing a tumor or cancer.

[0051] In another preferred embodiment, the first formulation and the second formulation are each independent.

[0052] In another preferred embodiment, the first formulation is a lyophilized formulation or a liquid formulation, preferably a liquid formulation.

[0053] In another preferred embodiment, the second formulation is a lyophilized formulation or a liquid formulation, preferably a liquid formulation.

[0054] In another preferred embodiment, the first formulation and the second formulation are each an injection.

[0055] In another preferred embodiment, the first formulation is administered before or after administration of the second formulation, or is administered simultaneously with the second formulation.

[0056] In a sixth aspect of the present application, a method for treating and / or preventing a disease is provided, the method comprising the step of administering to a subject in need thereof an inhibitor targeting SUCLG1 or a pharmaceutical composition according to the fourth aspect of the present application.

[0057] In another preferred embodiment, the disease comprises a tumor or cancer.

[0058] In another preferred embodiment, the tumor or cancer is a mitochondrial biogenesis-dependent tumor or cancer.

[0059] In another preferred embodiment, the mitochondrial biogenesis-dependent tumor or cancer comprises (but is not limited to) acute myeloid leukemia, colorectal cancer, liver cancer, lung cancer, etc.

[0060] In another preferred embodiment, the mitochondrial biogenesis-dependent tumor or cancer is acute myeloid leukemia, preferably FLT3-mutated acute myeloid leukemia.

[0061] In another preferred embodiment, the FLT3 mutation comprises a tyrosine kinase domain (TKD) mutation and an internal tandem duplication (ITD) mutation.

[0062] In another preferred embodiment, the subject in need thereof comprises a human or a non-human mammal.

[0063] In another preferred embodiment, the inhibitor targeting SUCLG1 is as previously described.

[0064] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 SUCLG1 was shown to maintain mtDNA-encoded gene expression and mitochondrial mass. Among others,

[0066] A. Spearman correlation of mitochondrial metabolism genes with ETC genes was analyzed in multiple TCGA transcriptomes.

[0067] B. Top ten mitochondrial metabolism genes positively correlated with ETC gene expression in the pan-cancer analysis.

[0068] C. shRNA to control or SUCLG1 was transduced into human cord blood (CB) cells or cancer cells, and mitochondrial mass was quantified by MTG staining; graphed as relative MTG fluorescence intensity compared to control.

[0069] D, E. Total RNA and total protein were extracted from control or SUCLG1 stably knocked down HL60 and MV411 cells. Relative mRNA expression of mtDNA and nDNA-encoded genes was quantified by qPCR and shown in log2 scale; protein expression was determined by Western blotting.

[0070] F, G. Transmission electron microscopy images of control and SUCLG1 knocked down MV411 cells were analyzed to determine the number of mitochondria in 100 cells (F). G shows representative images, scale bar: 1 pm. Data represent mean ± SD, n = 100 different cells, t test. **P < 0.01.

[0071] H-K. Wild-type SUCLG1 or its P170R (PR) and A209E (AE) mutants were re-expressed in SUCLG1 knocked down MV411 cells. Protein expression of mtDNA and nDNA-encoded genes was determined by Western blotting (H); and mtDNA abundance (I), mRNA expression levels (J) and oxygen consumption rate (K) were determined in stably cells. Data represent mean ± SD, n = 3 independent biological replicates, t test. **P < 0.01; n.s., not significant.

[0072] Figure 2 SUCLG1 was shown to limit POLRMT succinylation by reducing succinyl-CoA levels. Among others,

[0073] A. Schematic representation of the reactions catalyzed by the succinyl-CoA ligase complex. SDHA, succinate dehydrogenase subunit A; OGDH, alpha-ketoglutarate dehydrogenase; Pi, phosphate.

[0074] B. Metabolites were extracted from SUCLG1 knockdown and rescue MV411 cells and quantified by mass spectrometry. Relative abundance of metabolites shown in log2 scale.

[0075] C. Mitochondria were isolated from MV411 stable cell lines, metabolites were extracted to determine the abundance of succinyl-CoA. Data represent mean ± SD, n = 3 independent biological replicates, t-test. **P < 0.01.

[0076] D. Endogenous SUCLG1 was immunoprecipitated from MV411 cells, protein interactions of SUCLG1 were determined by Western blotting. IgG was used as negative control for immunoprecipitation. SUCLG1 knockdown cells were included to verify the specificity of the SUCLG1 antibody.

[0077] E, F. Endogenous POLRMT was immunoprecipitated from control or SUCLG1 knockdown MV411 (E) and HL60 (F) cells, lysine succinylation was determined by Western blotting. IgG was used as negative control for immunoprecipitation.

[0078] G. Immunopurified POLRMT-HA protein was incubated with succinyl-CoA (sucCoA) or succinate, immunopurified Flag-tagged SUCLG1 protein was treated or not with heat inactivation (95°C, 10 min), POLRMT succinylation was determined after in vitro succinylation.

[0079] H. Endogenous POLRMT was immunoprecipitated from SUCLG1 knockdown and rescue MV411 cells, lysine succinylation was determined by Western blotting. IgG was used as negative control for immunoprecipitation.

[0080] Figure 3 Succinylation of the lysine site at position 622 inhibits POLRMT activity. Among them,

[0081] A. Schematic representation of POLRMT structure. Cyan represents the POLRMT protein, brown represents the pentose phosphate backbone of the DNA-RNA hybridized strand.

[0082] B-E. Re-expression of HA-tagged POLRMT and its mutants in POLRMT knockdown MV411 cells, cells were further transfected with control or shRNA against SUCLG1. Immunopurification of POLRMT-HA to determine its succinylated and protein interactions (B). MitoChIP was performed to assess the binding of POLRMT-HA to mtDNA (C). Mitochondria were isolated to label assess the rate of nascent RNA (D). UTP label amount was quantified and normalized to mtDNA (E). Data represent mean ± SD, n = 3 independent biological replicates, one-way ANOVA, Dunnett’s multiple comparison test. **P < 0.01; n.s., not significant.

[0083] F-H. Determination of relative mtDNA abundance (F), mRNA expression levels of mtDNA-encoded genes (G) and oxygen consumption rate (H) in MV411 stable cell lines. Data represent mean ± SD, n = 3 independent biological replicates, t-test. *P < 0.01; **P < 0.01; n.s., not significant.

[0084] Figure 4 It is shown that mutated FLT3 upregulates SUCLG1 to promote mitochondrial biogenesis in leukemia. Among others,

[0085] A. Protein expression was determined by Western blotting in different AML cells. CD34+ CB cells from three different donors were used as normal controls, K622 succinylation (K622sc) was detected on immunopurified endogenous POLRMT. AML cell lines carrying FLT3 mutations are marked in red. IgG was used as negative control for immunoprecipitation. The ratio of K622sc and SUCLG1 was normalized to POLRMT protein and Actin, respectively.

[0086] B, C. Metabolites were extracted to determine cellular succinyl-CoA levels. Correlation of POLRMT K622sc with SUCLG1 protein (B) and succinyl-CoA levels (C) was analyzed.

[0087] D-H. HA-tagged FLT3 and its mutants were stably expressed in CD34+ CB cells, endogenous POLRMT was immunopurified to determine K622 succinylation. The ratio of K622sc was normalized to POLRMT protein (D). Cellular succinyl-CoA levels (E), mtDNA abundance (F), mtDNA-encoded genes expression (G) and MTG intensity (H) were determined. Data represent mean ± SD, n = 3 independent biological replicates, t-test. **P < 0.01; n.s., not significant.

[0088] I-M. shRNA against FLT3 was stably expressed in MV411 cells, endogenous POLRMT was immunopurified to determine K622 succinylation. The ratio of K622sc was normalized to POLRMT protein (I). Cellular succinyl-CoA levels (J), mtDNA abundance (K), mtDNA-encoded gene expression (L) and MTG intensity (M) were determined. Data represent mean ± SD, n = 3 independent biological replicates, t test. **P < 0.01; n.s., not significant.

[0089] Figure 5 SUCLG1 was shown to promote the development of leukemia by inhibiting the succinylation of POLRMT.

[0090] wherein,

[0091] A. Proliferation experiments of SUCLG1 knockdown and -complemented MV411 cells. Data represent mean ± SD, n = 3 independent biological replicates, t test. **P < 0.01; n.s., not significant.

[0092] B, C. SUCLG1 knockdown and -complemented cells were treated with a POLRMT inhibitor (POLRMTi, IMT1 (MedChemExpress # HY-134539)) and subjected to cell proliferation (B) and colony formation (C) experiments. Data represent mean ± SD, n = 3 independent biological replicates, t test. *P < 0.05; **P < 0.01; n.s., not significant.

[0093] D, E. SUCLG1 knockdown and -complemented cells were treated with FLT3 inhibitor FLT-IN-3 and subjected to cell proliferation (D) and colony formation (E) experiments. Data represent mean ± SD, n = 3 independent biological replicates, t test. *P < 0.05; **P < 0.01; n.s., not significant.

[0094] F. Mice MLL-AF9+FLT3 ITD Schematic representation of leukemia model establishment.

[0095] G, H. MLL-AF9 bone marrow cells were transfected with control vector or HA-tagged FLT3 ITD , stably expressing control or shRNA against Suclg1. Protein expression was determined by Western blotting (G). Animal survival experiments were performed after transplantation of leukemia bone marrow (H). n = 9 mice, log-rank (Mantel-Cox) test. **P < 0.01.

[0096] I. POLRMT knockdown and rescue MV411 cells were transfected with either a blank control or shRNA against SUCLG1, and cells were transplanted into sublethally irradiated NSG mice to determine animal survival. n = 5 mice, log-rank (Mantel-Cox) test. **P < 0.01. J-L Human clinical AML bone marrow samples were collected. Metabolites were extracted to determine the level of succinyl-CoA (J). The K622 succinylation of immunopurified POLRMT (K) and SUCLG1 protein expression (L) were determined by Western blotting. n = 5-10, t-test. *P < 0.05; **P < 0.01; n.s., not significant.

[0097] M-N. FLT3 WT (n = 116) and FLT3 TKD / ITD (n = 38) TCGA AML samples. t-test. *P < 0.05; **P < 0.01; n.s., not significant. TCGA AML patients were grouped according to FLT3 mutational status and SUCLG1 mRNA expression level. O. Overall survival of AML samples was determined. log-rank (Mantel-Cox) test. *P < 0.05; n.s., not significant.

[0098] P. Schematic diagram of the working model of SUCLG1 -mediated succinyl-CoA signaling and FLT3 -induced remodeling of mitochondrial biogenesis. DETAILED DESCRIPTION

[0099] The present inventors, through extensive and in-depth research, first accidentally found that the tricarboxylic acid cycle enzyme succinyl-CoA synthetase SUCLG1 is closely related to the expression of a variety of mitochondrial electron transport chain (ETC) genes in the TCGA cancer transcriptome, and further found that SUCLG1 limits the level of succinyl-CoA, inhibits the succinylation of the 622nd lysine site of mitochondrial RNA polymerase (POLRMT) to maintain mtDNA transcription, mitochondrial biogenesis and tumor cell proliferation. In particular, for mitochondrial biogenesis-dependent tumors (such as acute myeloid leukemia, especially FLT3 mutant acute myeloid leukemia), SUCLG1 plays an important role in the process of tumor progression. Therefore, the applicant proposes that inhibiting SUCLG1 can promote the succinylation of the POLRMT protein, thereby disrupting the interaction of POLRMT with mtDNA and mitochondrial transcription factors, reducing mitochondrial biogenesis, and thus limiting tumor progression. And the role of inhibiting SUCLG1 in limiting tumor progression is verified by in vivo and in vitro experiments, providing a new target for tumor treatment. On this basis, the present application is completed.

[0100] Terms

[0101] succinyl-CoA synthetase

[0102] Succinyl-CoA synthetase (SUCLG1) is a mitochondrial enzyme that catalyzes the combination of succinate and coenzyme A to form succinyl-CoA, a key step in the tricarboxylic acid cycle. Specific information about the succinyl-CoA synthetase (SUCLG1) referred to in the present invention can be found in NCBI: NP_003840.2.

[0103] Mitochondrial genome

[0104] Mitochondrial DNA is the genetic material in mitochondria, also known as mtDNA, which exists independently of the nuclear genome. Mutations in mtDNA-encoded genes can cause mitochondrial diseases. mtDNA has a mitochondria-specific replication, transcription mechanism.

[0105] Mitochondrial RNA polymerase

[0106] Mitochondrial DNA transcription RNA polymerase (POLRMT) is encoded by nuclear DNA and synthesized on cytoplasmic ribosomes, and then transported to mitochondria to be responsible for mtDNA transcription and replication. Specific information about the mitochondrial RNA polymerase (POLRMT) referred to in the present invention can be found in NCBI: NP_001394734.1.

[0107] Mitochondrial biogenesis

[0108] As used herein, the terms "mitochondrial biogenesis", "mitogenesis" can be used interchangeably. Mitochondrial biogenesis refers to the process of formation and expansion of mitochondria, including mitochondrial genome replication, gene transcription and mitochondrial protein synthesis, in addition to the biosynthesis of other mitochondrial components such as lipids and respiratory chain components.

[0109] As used herein, the term "abnormal regulation of mitochondrial biogenesis" includes: enhancement or attenuation of mitochondrial biogenesis in tumor cells, epigenetic modification and transcriptional disorder of mitochondrial genes, abnormal regulation of mitochondrial protein expression and abnormality of mitochondrial activity.

[0110] Active ingredient combination, pharmaceutical composition of the present invention

[0111] The present invention provides an active ingredient combination comprising a first active ingredient targeting an inhibitor of SUCLG1, and a second active ingredient targeting an inhibitor of POLRMT.

[0112] Based on the above combination, the present invention provides a pharmaceutical composition which can be used for: reducing mitochondrial biogenesis; and / or treating or preventing a mitochondrial biogenesis-dependent tumor or cancer.

[0113] The pharmaceutical composition of the present application comprises: (a) an inhibitor targeting SUCLG1, (b) an inhibitor targeting POLRMT, and (c) a pharmaceutically acceptable carrier.

[0114] Generally, the inhibitor targeting SUCLG1 and / or the inhibitor targeting POLRMT of the present application can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, wherein the pH is generally about 5-8, preferably, the pH is about 6-8.

[0115] As used herein, the term "inhibitor targeting SUCLG1" refers to an agent capable of down-regulating the expression of SUCLG1 gene (including transcriptional level and protein level), and / or inhibiting or blocking the functional activity of SUCLG1, including (but not limited to) small molecule compounds, polypeptides, nucleic acids (e.g., shRNA, siRNA or sgRNA). After the inhibitor targeting SUCLG1 is administered to a subject, it reduces mitochondrial biogenesis by promoting the succinylation modification of mitochondrial RNA polymerase (POLRMT), disrupting the binding of POLRMT to mitochondrial transcription factors TFAM and TFB2M, and disrupting the binding of POLRMT to mtDNA, thus having a significant anti-tumor effect on tumors or cancers that are dependent on mitochondrial biogenesis. In a preferred embodiment of the present application, the inhibitor targeting SUCLG1 is an shRNA targeting SUCLG1.

[0116] As used herein, the term "inhibitor targeting POLRMT" refers to an agent capable of down-regulating the expression of POLRMT gene (including transcriptional level and protein level), or capable of inhibiting or blocking the activity of POLRMT, including (but not limited to) small molecule compounds, polypeptides, nucleic acids (e.g., shRNA, siRNA or sgRNA). After the inhibitor targeting POLRMT is administered to a subject, it can inhibit mtDNA replication and transcription, reducing mitochondrial biogenesis. In a preferred embodiment of the present application, the inhibitor targeting POLRMT is an shRNA targeting POLRMT. In another preferred embodiment of the present application, the inhibitor targeting POLRMT is a POLRMT-specific chemical inhibitor (referred to as POLRMTi in the present application). In a specific embodiment of the present application, the inhibitor targeting POLRMT is IMT1 (MedChemExpress #HY-134539).

[0117] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

[0118] The term "pharmaceutically acceptable carrier" refers to a carrier that is not itself toxic and does not significantly interfere with the activity of the active ingredient. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition can include liquids such as water, saline, buffers. In addition, these carriers can also contain auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, etc. The carriers can also contain cell transfection reagents.

[0119] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in a human and / or animal and / or cell and can be accepted by the human and / or animal.

[0120] As used herein, "pharmaceutically acceptable" refers to those substances that are suitable for use in humans and / or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio, i.e., substances that are acceptable to the U.S. Food and Drug Administration as well as the European Medicines Agency. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of therapeutic agents, including various excipients and diluents. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical preparation of the present application can be prepared in the form of a needle injection, for example, by a conventional method using physiological saline or an aqueous solution containing dextrose and other auxiliary agents. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical composition of the present application can also be prepared as a sustained-release preparation.

[0121] When using the pharmaceutical preparation, a safe and effective amount of the active ingredient combination (inhibitors targeting SUCLG1 and / or inhibitors targeting POLRMT) is administered to the mammal.

[0122] It should be understood that the effective amount of the active ingredient of the present application can vary depending on the mode of administration and the severity of the disease, etc. The selection of the preferred effective amount can be determined by a person skilled in the art according to various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; severity of the disease, body weight of the patient, immune status of the patient, route of administration, etc.

[0123] The mode of administration of the pharmaceutical composition of the present application is not particularly limited, and representative examples include, but are not limited to, intravenous injection, intravenous infusion, intratumoral injection, subcutaneous injection, intramuscular injection, etc.

[0124] Use of the present application

[0125] The present application provides the use of an inhibitor targeting SUCLG1 for the preparation of a composition or a medicament for promoting the succinylated modification of mitochondrial RNA polymerase (POLRMT), inhibiting the activity of POLRMT, thereby reducing mitochondrial biogenesis; and / or treating or preventing a tumor or a cancer.

[0126] Further, the present application also provides the use of an active ingredient combination for the preparation of a composition or a medicament for reducing mitochondrial biogenesis; and / or treating or preventing a tumor or a cancer dependent on mitochondrial biogenesis, said combination comprising a first active ingredient, an inhibitor targeting SUCLG1, and a second active ingredient, an inhibitor targeting POLRMT.

[0127] The inhibitor targeting SUCLG1 and the active ingredient combination according to the present application are particularly suitable for use in a tumor or a cancer, the occurrence and / or progression of which is particularly associated with abnormal regulation of mitochondrial biogenesis, or is referred to as a mitochondrial biogenesis-dependent tumor. Thus, the inhibitor targeting SUCLG1 and the active ingredient combination according to the present application are particularly suitable for use in the treatment or prevention of acute myeloid leukemia (AML), colorectal cancer, liver cancer, lung cancer, etc., in particular acute myeloid leukemia (AML). In addition, it has been found by the present application that mutant FLT3 upregulates SUCLG1 to remodel mitochondrial biogenesis and promote the progression of AML, and thus the inhibitor targeting SUCLG1 and the active ingredient combination according to the present application are particularly suitable for use in the treatment or prevention of FLT3 mutant acute myeloid leukemia (AML).

[0128] The advantages of the present application include:

[0129] (1) The present application first found that SUCLG1 is closely related to the expression of mitochondrial electron transport chain (ETC) genes in the cancer transcriptome.

[0130] (2) The present application first established the regulatory relationship between SUCLG1 and intracellular metabolism, mitochondrial genome (mtDNA).

[0131] (3) The present application first revealed the intrinsic mechanism of SUCLG1 regulating mitochondrial biogenesis: SUCLG1 limits the level of succinyl-CoA, inhibits the succinylation of the lysine site at position 622 of mitochondrial RNA polymerase (POLRMT), thereby maintaining mtDNA transcription and mitochondrial biogenesis.

[0132] (4) The present application first proposes and verifies that targeted inhibition of SUCLG1 can promote the succinylation of POLRMT, thereby disrupting the interaction of POLRMT with mtDNA and mitochondrial transcription factors, reducing mitochondrial biogenesis, and thus limiting tumor progression. Moreover, targeted inhibition of SUCLG1 has a significant tumor inhibitory effect on mitochondrial biogenesis-dependent tumors (such as acute myeloid leukemia, especially FLT3-mutated acute myeloid leukemia). Therefore, a new target is provided for tumor treatment.

[0133] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional regulations, such as the conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (Fourth Edition) (Science Press Limited Liability Company, 2017), or according to the conditions described in the product instruction manual provided by the product manufacturer. The materials, reagents, instruments, etc. used in the examples, if not specifically stated, can be obtained from commercial channels.

[0134] Experimental materials and methods

[0135] Cells, antibodies and tumor samples

[0136] All cells were cultured at 37°C in a 5% C02humidified environment. Human embryonic kidney cell line (HEK293T), human AML cell lines (HL60, KG1, MOLM14, MONOMAC6, MV411, NB4, NOMO1, THP1), colon cancer cell line (HCT116), lung cancer cell line (A549) and liver cancer cell line (HepG2) were cultured in RPMI 1640 medium or Dulbecco's modified Eagle medium (DMEM) (Invitrogen) containing penicillin, streptomycin and 2 mM glutamine (Corning) and 10% fetal bovine serum (Biological Industries). Human cord blood CD34+ cells were cultured in RPMI 1640 medium containing recombinant human growth factors including 40 ng / mL IL-6 (Peprotech), 50 ng / mL FLT3 ligand (Peprotech), 20 ng / mL stem cell factor (SCF) (Peprotech) and 50 ng / ml TPO (Peprotech). Mouse bone marrow cells were cultured in RPMI 1640 medium containing recombinant mouse growth factors including 10 ng / mL IL3 (R&D systems), 10 ng / mL stem cell factor (SCF) (R&D systems), 100 ng / mL IL6 (Peprotech). Cells were treated with SIRT5 inhibitor (SIRT5i, 0.5 mM, MedChemExpress #HY-112634) or FLT3-IN-3 (1 nM, MedChemExpress #HY-112145) for the indicated times.

[0137] cDNAs encoding human SUCLG1, POLRMT and FLT3 were cloned into Flag, HA or His tagged vectors (pcDNA3.1, pSJ3 and pLV-EFla-IRES). All expression constructs were verified by DNA sequencing. Plasmid transfection was performed using polyethylenimine or Lipofectamine 3000 (ThermoFisher). Commercially purchased antibodies include MT-COl (Novus biologicals #NBP2-29949), MT-ND6 (Novus biologicals #NBP1-70650), MT-ATP6 (Sigma #MABS1995), SDHA (Cell Signaling #11998), NDUFB8 (Novus biologicals #NBP2-75586), POLG (Abeam #abl28899), POLRMT (Abeam #abl67368), MRPL45 (Sigma #HPA023373), PGC-l a (Abeam #ab54481), NRF2 (Abeam #ab89443), b-actin (Cell Signaling #3700), Flag (Sigma #F1804), SUCLG1 (Abeam #ab97867), TFAM (Cell Signaling #8076), suc-Lys (Novus biologicals #NBP3-16032), HA (Santa Cruz #sc-7392), HSP60 (Cell Signaling #4870), G6PD (Abeam #abl35525), Histone H3 (Cell Signaling #3638), me-Lys (Cell Signaling #14679), ac-Lys (Cell Signaling #9441), p-Tyr (Santa Cruz #sc-508), SUCLG2 (Santa Cruz #sc-390818), SUCLA2 (Novus biologicals #NBP1-33015), His (Cell Signaling #2366), TFB2M (Cell Signaling #50837), FLT3 (Cell Signaling #3462).

[0138] AML samples were derived from 15 AML cases from Chongqing Southwest Hospital.

[0139] shRNAs targeting SUCLG1 / POLRMT and SUCLG1 / POLRMT expression knockdown Stable cell lines were constructed for shRNA cloning using pMKO.1-puro, pMKO.1-hygro and pLKO.1-blast vectors. Cells stably knocked down for genes were constructed using shRNAs against SUCLG1, POLRMT and FLT3. Retroviruses were prepared by using a two-plasmid packaging system (gag and vsvg). Cells were infected with 8 pg / mL polybrene and the virus solution after centrifugation. After one week of selection with puromycin (4 pg / mL) or hygromycin (200 pg / mL), stable knockdown cells were obtained.

[0140] To generate stable cell lines for SUCLG1 expression knockdown and rescue, Flag-tagged wild-type SUCLG1 or its mutants were cloned into lentiviral pLV-EF1a-IRES-puro vector and co-transfected with a vector expressing gag and vsvg genes in HEK293T cells to produce lentivirus. Cells used for transduction stably expressed shSUCLG1-#1 or shSUCLG1-#2 targeting 3'UTR. After one week of selection with puromycin (4 pg / mL).

[0141] To generate stable cell lines for POLRMT knockdown and rescue, HA-tagged human wild-type POLRMT or its mutants containing two silent nucleotide substitutions in the sequence corresponding to the shRNA targeting region were cloned into lentiviral pLV-EF1a-IRES-puro vector and co-transfected with a vector expressing gag and vsvg genes in HEK293T cells to produce lentivirus. Cells used for transduction stably expressed shPOLRMT-#1. After one week of selection with puromycin (4 pg / mL).

[0142] Nucleotide sequence of shSUCLG1-#1:

[0143] Forward: ccggTGGAATGGATCACGTAGACATctcgagATGTCTACGTGATCCATTCCAtttttg (SEQ ID NO: 1)

[0144] Reverse: aattcaaaaaTGGAATGGATCACGTAGACATctcgagATGTCTACGTGATCCATTCCA (SEQ ID NO: 2)

[0145] Nucleotide sequence of shSUCLG1-#2:

[0146] Forward:

[0147] ccggGCATCATGCCTGGCCATATTCctcgagGAATATGGCCAGGCATGATGCtttttg (SEQ ID NO: 3)

[0148] Reverse: aattcaaaaaGCATCATGCCTGGCCATATTCctcgagGAATATGGCCAGGCATGATGC (SEQ ID NO: 4)

[0149] Nucleotide sequence of shPOLRMT-#1:

[0150] Forward: ccggGACTCCAAGGTCAAGCAAATActcgagTATTTGCTTGACCTTGGAGTCtttttg (SEQ ID NO: 5)

[0151] Reverse: aattcaaaaaGACTCCAAGGTCAAGCAAATActcgagTATTTGCTTGACCTTGGAGTC (SEQ ID NO: 6)

[0152] Specific antibodies for succinylated K622 (anti-K622sc) and their preparation

[0153] To generate site-specific antibodies for detecting POLRMT succinylation [a-K622sc], the synthetic polypeptide QIGILK(sc)PHPAY (GL Biochem) was coupled to KLH as an anti-rabbit antigen. After four immunizations, antisera were collected.

[0154] In vitro succinylation experiments

[0155] Purified His-tagged POLRMT in bacteria was co-incubated with different concentration gradients of succinyl-CoA or succinate in the presence or absence of immunopurified SUCLG-Flag. The in vitro succinylation reaction buffer consisted of 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. The mixture was incubated at 37°C for 60 min. The products were detected for succinylation by Western Blotting.

[0156] Mitochondrial genome immunoprecipitation (MitoChIP)

[0157] Cells were resuspended in mitochondrial isolation buffer (MIB) [containing 310 mM sucrose, 10 mM Tris-HCl (pH 7.5) and 0.05% BSA (w / v), supplemented with protease inhibitor cocktail (Sigma)], ground, and centrifuged at 1000 g for 10 min at 4°C. The supernatant was further centrifuged at 4500 g for 15 min at 4°C, and the pellet was washed once with MIB. The crude mitochondrial pellet was resuspended in MIB containing protease inhibitors. Next, the isolated mitochondria were cross-linked with 1% formaldehyde and sonicated. The disrupted mitochondrial chromatin was immunoprecipitated using antibodies against HA, POLRMT, and control IgG. The antibody-chromatin complexes were precipitated by protein A agarose (Millipore), washed, and eluted. After cross-linking reduction and proteinase K treatment, the immunoprecipitated DNA was extracted with phenol-chloroform and precipitated in ethanol. The DNA fragments were further analyzed by qPCR.

[0158] Extraction and analysis of cellular metabolites

[0159] Approximately 5 x 105 6 Cells were washed in 1 mL of physiological saline (0.9% NaCl solution) and lysed in ice-cold methanol (HPLC grade). Water and chloroform were added to the lysate (volume ratio methanol:water:chloroform = 2:1:2), followed by shaking and centrifugation (5000 g, 4°C, 15 min). The aqueous phase was evaporated dry by a nitrogen stream at 37°C. The extracted metabolites were resuspended in 50% acetonitrile and subjected to mass spectrometry analysis. The relative metabolite content was calculated by normalizing the relative metabolite abundance to the total protein amount.

[0160] Statistical analysis

[0161] The relative concentration of immunoprecipitated proteins was quantified by measuring the Western Blotting band intensity by ImageQuant TL (Amersham Biosciences). Spearman correlation data of mitochondrial metabolic genes with electron transport chain (ETC) genes were available from cBioPortal (https: / / www.cbioportal.org / ). t-test or One-way ANOVA with Dunnett's multiple comparison test were performed using GraphPad Prism to determine statistical significance. Survival of leukemia patients was plotted by Kaplan-Meier survival curves and analyzed using the log-rank (Mantel-Cox) test. All data shown represent the results of at least three independent experiments. p-values < 0.05 were considered statistically significant.

[0162] Example 1 SUCLG1 maintains mtDNA-encoded gene expression and mitochondrial mass

[0163] To determine which metabolic enzymes are involved in regulating mtDNA function, the present inventors analyzed the correlation between mitochondrial metabolic genes and ETC genes in TCGA transcriptome data ( Figure 1 According to MitoCarta3.0 (Rath et al, 2021), 397 of the 405 mitochondrial genes involved in metabolism had valid Spearman correlations with ETC genes in 32 different cancer types in the TCGA firehose legacy dataset (Gao et al, 2019). The cancer-specific ranking of ETC correlation coefficients showed that SUCLG1 was strongly correlated with ETC gene expression in various cancers ( Figure 1 AB). SUCLG1 converts succinyl-CoA into succinate, and both metabolites are important signaling molecules in cellular metabolism. Acute myeloid leukemia (AML) cells were selected as a model because they are highly dependent on mitochondrial respiration. In the TCGA AML dataset, the ranking of mitochondrial enzymes showed that the metabolic enzyme DGUOK, which provides precursors for mtDNA synthesis, was most significantly correlated with ETC (Mandel et al., 2001). This observation supports the reliability of the above correlation analysis. Next, we explored whether SUCLG1 acts as a positive regulator of mitochondrial development. To verify this, two different short hairpin RNAs (shRNAs) that downregulated SUCLG1 expression were stably expressed in human leukemia cell lines (HL60, THP1, KG1, MOLM14, MONOMAC6, MV411); at the same time, CD34+ cord blood cells (CD34 + CB) was used as a normal control. Cells were stained with MitoTracker Green (MTG), a specific dye for labeling mitochondria. As expected, downregulation of SUCLG1 expression significantly reduced the abundance of mtDNA and MTG intensity in cells ( Figure 1 Furthermore, downregulation of SUCLG1 expression also reduced mitochondrial mass in colorectal cancer (HCT116), lung cancer (A549), and liver cancer (HepG2) cell lines ( Figure 1 Middle C). In summary, SUCLG1 positively regulates mitochondrial mass.

[0164] Since SUCLG1 is located in the mitochondrial matrix, we speculated that SUCLG1 may regulate the biogenesis process within mitochondria. Supporting this view, the reduction of SUCLG1 in AML cell lines (HL60 and MV411) reduced the mRNA and protein expression of mtDNA-encoding genes, while the expression of key regulators of mtDNA replication, transcription, and protein translation: DNA polymerase γ (POLG), POLRMT, and mitochondrial ribosomal protein L45 (MRPL45) was largely unchanged; in addition, nuclear factors that control mitochondrial biogenesis, including peroxisome proliferator-activated receptor γ coactivator 1α (PGC1-α) and nuclear factor erythroid 2-related factor 2 (NRF2), showed slight changes ( Figure 1 These results suggest that SUCLG1 maintains the expression of mtDNA-encoded genes. To verify the role of SUCLG1 in mitochondrial biogenesis, electron microscopy was used to quantify the number of mitochondria in MV411 cells. Consistent with previous results, the number of mitochondria in SUCLG1-knockdown cells was reduced by approximately 50% ( Figure 1 These data collectively support a role for SUCLG1 in maintaining mitophagy.

[0165] Congenital SUCLG1 mutations in humans are associated with clinical mtDNA depletion syndrome through unknown mechanisms (Ostergaard et al., 2007). The disease-associated mutation sites, proline 170 (P170) and alanine 209 (A209) residues, are highly conserved during evolution and are located near the catalytic site of SUCLG1 (Huang & Fraser, 2021). Therefore, disease-associated SUCLG1 mutants P170R and A209E with Flag tags were constructed (Chinopoulos et al, 2019; Rouzier et al, 2010) to test their biological functions. Enzyme activity assays using immunopurified SUCLG1-Flag proteins showed that P170R and A209E mutants showed a loss of enzymatic activity compared to the wild type. In addition, SUCLG1 P170R and SUCLG1 A209E Re-expression into SUCLG1 knockdown MV411 cells ( Figure 1 Middle H). SUCLG1 mutants fail to restore mtDNA abundance, expression of mtDNA-encoded genes, and cellular respiration ( Figure 1 These data indicate that SUCLG1 activity is essential for maintaining mitochondrial mass.

[0166] Example 2 SUCLG1 inhibits succinylation of POLRMT by reducing succinyl-CoA levels

[0167] Next, we investigated how SUCLG1 regulates the organelle-specific mitochondrial biogenesis mechanism. Succinyl-CoA synthetase is a heterodimeric enzyme in which SUCLG1 binds to SUCLG2 or SUCLA2 to convert succinyl-CoA to succinate, while generating GTP or ATP, respectively (Fig. 1A). Succinyl-CoA is produced from a-ketoglutarate (a-KG) by a-ketoglutarate dehydrogenase (OGDH) (Fig. 1A). Unlike SUCLG1, SUCLG2, and SUCLA2, OGDH did not show a significant correlation with ETC gene expression in the TCGA transcriptome. Thus, we focused on how SUCLG1 links metabolism to mitochondrial biogenesis. Metabolic product profiling showed that knocking down SUCLG1 in MV411 cells increased the levels of a-KG and succinyl-CoA, accompanied by decreased ATP and GTP levels; re-expression of wild-type SUCLG1, but not the inactive deletion mutant SUCLG1, restored the abundance of succinyl-CoA (Fig. 1B). Since succinyl-CoA is localized in a regional manner in mitochondria, we further isolated mitochondria to quantify succinyl-CoA. The results showed a similar change pattern in mitochondria as in cells (Fig. 1C). Taken together, SUCLG1 inhibits succinyl-CoA in mitochondria, which can contribute to its role in regulating mitochondrial biogenesis. Figure 2 Figure 2 Figure 2 Figure 2

[0168] Succinyl-CoA acts as a succinyl donor in the succinylation reaction to modify substrate proteins. We thus hypothesized that SUCLG1 can modify the succinylation of proteins. To identify potential targets of SUCLG1, we examined the interaction between SUCLG1 and mitochondrial DNA replication and transcription regulators. Interestingly, endogenous SUCLG1 interacted with POLRMT and TFAM, but not with POLG, in MV411 cells (Fig. 1D). POLRMT directly mediates mitochondrial DNA transcription. In SUCLG1 expression-knocked-down MV411 and HL60 cells, the level of endogenous POLRMT succinylation was increased (Fig. 1E-F). These results suggest that SUCLG1 limits the succinylation of POLRMT. Figure 2 Figure 2

[0169] ​​​​​​Succinylation occurs non-enzymatically, allowing mitochondrial succinyl-CoA levels to be directly linked to the succinylation of target proteins. To determine whether succinyl-CoA acts as a metabolic signal to modify POLRMT, HA-tagged POLRMT and Flag-tagged SUCLG1 were immunopurified from MV411 cells and pre-incubated with SUCLG1-Flag protein in reaction buffer for in vitro succinylation reactions. In vitro succinylation reactions revealed that the SUCLG1-Flag enzyme inhibited succinylation of POLRMT, whereas its heat-inactivated form (HI) had no such effect ( Figure 2 G). It can be seen that SUCLG1 limits the succinylation of POLRMT induced by succinyl-CoA in vitro. In addition, POLRMT was immunopurified from MV411 stable cells with SUCLG1 knockdown, and its succinylation modification level was detected. The results showed that re-expression of wild-type SUCLG1, but not its mutant, restored the lysine succinylation of endogenous POLRMT ( Figure 2 In summary, SUCLG1 downregulates succinylation of POLRMT by limiting succinyl-CoA levels.

[0170] Example 3 Succinylation modification at K622 inhibits the activity of POLRMT

[0171] Further investigation of the biological function of POLRMT succinylation. Proteomic analysis showed that lysine 622 (K622) of POLRMT is a conserved site where succinylation may occur (Weinert et al, 2013). K622 is located in the DNA binding region of POLRMT (Schwinghammer et al, 2013) ( Figure 3 (A) Succinylation at K622 may affect POLRMT binding to mtDNA due to steric hindrance, thereby affecting mtDNA transcriptional activity. To accurately detect succinylation of POLRMT, a specific antibody against succinylated K622 (anti-K622sc) was prepared. K622 was also mutated to arginine (K622R) or glutamic acid (K622E), mimicking its unmodified and succinylated states, respectively (Qi et al, 2019). HA-tagged POLRMT and its mutants were immunopurified, and the anti-K622sc antibody specifically recognized wild-type POLRMT but not the K622R or K622E mutants. Sirtuin 5 (SIRT5) is a mitochondrial deacylase responsible for removing succinylation modifications. Inhibition of SIRT5 with specific small molecules significantly increased the succinylation level at K622 of wild-type POLRMT, but not its mutants. These results indicate that K622 is the major succinylation site of POLRMT.

[0172] To determine the physiological function of POLRMT succinylation, wild-type POLRMT with HA tag and its mutants (POLRMT K622R and POLRMT K622E ) were re-expressed in POLRMT knockdown MV411 and HL60 cells. Co-immunoprecipitation experiments showed that POLRMT K622R interacted more strongly with TFAM and TFB2M than wild-type POLRMT. In contrast, POLRMT K622E mutants with mimicked succinylation had a poor ability to bind to mitochondrial transcription factors ( Figure 3 B in the middle). Furthermore, shRNA targeting SUCLG1 was expressed in POLRMT expression knockdown and complemented stable cells. Knockdown of SUCLG1 led to over-succinylation of immunopurified POLRMT, accompanied by a decreased ability of POLRMT to bind to TFAM and TFB2M ( Figure 3 B in the middle). These results suggest that SUCLG1 limits the succinylation of K622 site of POLRMT to maintain the interaction of the transcriptional complex.

[0173] The sugar-phosphate backbone of DNA carries a negative charge, which provides a structural basis for K622-mediated binding of POLRMT to mtDNA. The present inventors speculate that succinylation of K622 site will disrupt the binding of POLRMT to mtDNA and inhibit mitochondrial transcription. To verify this, a mitochondrial genome immunoprecipitation (MitoChIP) experiment was performed to determine the interaction of POLRMT with mtDNA. The results showed that wild-type POLRMT and its K622R mutant interacted significantly with mtDNA, and the binding ability of POLRMT K622E with mimicked succinylation to mtDNA was impaired ( Figure 3 C in the middle). Importantly, in SUCLG1 expression knockdown cells, the mtDNA binding of wild-type POLRMT was reduced, but its K622 mutant was not affected by the binding to mtDNA ( Figure 3 C in the middle). Next, an in vitro transcription experiment was performed to directly monitor the activity of POLRMT using radiolabeled UTP. Compared with wild-type POLRMT, the K622E mutant failed to maintain UTP radiolabeling in mitochondria in MV411 stable cells ( Figure 3 D-E in the middle). Knockdown of SUCLG1 significantly reduced mitochondrial transcription in POLRMT WT complemented cells, but had a weaker effect in K622E mutant complemented cells ( Figure 3 D-E in the middle). Similarly, SUCLG1 expression knockdown effectively reduced the expression of POLRMTWT mtDNA content, mtDNA-encoded gene expression, and oxygen consumption in the cells were significantly decreased, but the decrease was less in cells complemented with the K622 mutant ( Figure 3 Together, these results demonstrate that SUCLG1 maintains mitochondrial transcription and respiration by inhibiting succinylation at K622.

[0174] Example 4 FLT3 mutants in AML cells upregulate SUCLG1 to promote mitochondrial biogenesis

[0175] AML (acute myeloid leukemia) is a genetically heterogeneous malignancy with active mitochondrial biogenesis, resulting in an increased dependence on mitochondrial respiration. To identify genetic factors that remodel mitochondrial biogenesis in AML, a panel of AML cell lines was tested for POLRMT succinylation ( Figure 4 Human CD34+ cord blood cells from three different healthy donors were used as normal controls. Endogenous POLRMT was immunoprecipitated from various cell lines, and succinylation of K622 was determined by western blotting. Interestingly, SUCLG1 protein expression in AML cell lines was negatively correlated with the level of succinylation of K622 ( Figure 4 Further metabolites were extracted from the cell line and the intracellular succinyl-CoA content was quantified. It was found that the succinyl-CoA content was positively correlated with POLRMT succinylation ( Figure 4 Middle C).

[0176] FLT3 is a membrane-bound tyrosine kinase that is mutated in approximately 30% of AML patients. Notably, SUCLG1 is overexpressed in FLT3-mutant cell lines including MONOMAC6, MOLM14, and MV411; accordingly, the succinylation level of K622 is lower in FLT3-mutant cells compared with other AML cell lines and healthy controls ( Figure 4 Middle A). These results suggest that FLT3 may remodel mitochondrial biogenesis in AML cells.

[0177] Mutations in the tyrosine kinase domain (TKD) or internal tandem duplication (ITD) of FLT3 lead to its constitutive activation. To test whether FLT3 regulates mitochondrial biogenesis, HA-tagged FLT3 and its mutants were overexpressed in CD34+ cord blood cells. TKD and FLT3 ITD Mutant, but not wild-type, FLT3, enhanced SUCLG1 expression and decreased succinyl-CoA levels ( Figure 4 Meanwhile, overexpression of FLT3 mutant reduced the succinylation level of POLRMT ( Figure 4Mid D), mtDNA content, mtDNA-encoded gene expression, mitochondrial mass and cellular respiration were significantly increased Figure 4 Mid F-H). Moreover, knockdown of FLT3 expression or inhibition of FLT3 activity downregulated SUCLG1 expression, increased succinyl-CoA levels and the level of succinylation at K622 site of POLRMT in MV411 and MOLM14 cells Figure 4 Mid I-J). More importantly, FLT3 knockdown downregulated mtDNA content, mtDNA-encoded gene expression and mitochondrial mass in MV411 cells Figure 4 Mid K-M). These results suggest that FLT3 mutants upregulate SUCLG1 to inhibit the succinylation of POLRMT, thereby enhancing mitochondrial biogenesis in leukemia cells.

[0178] Example 5 SUCLG1 inhibits the succinylation of POLRMT to promote leukemia cell proliferation

[0179] Given the importance of mitochondrial respiration for leukemia cell proliferation, AML was chosen as a model to study the pathophysiological function of POLRMT succinylation. Knockdown of SUCLG1 inhibited the proliferation of MV411 cells, and WT restored the proliferation of cells, but back-supplementation of its catalytic mutant failed to restore cell proliferation Figure 5 Mid A). POLRMT-specific inhibitors significantly inhibited the proliferation of SUCLG1 WT back-supplemented cells and clonogenic formation, but had no further inhibitory effect on the proliferation of cells back-supplemented with SUCLG1 mutants Figure 5 Mid B-C). Similar results were observed when SUCLG1 knockdown and back-supplemented cells were treated with FLT-IN-3, an inhibitor of FLT3 Figure 5 Mid D-E). These data suggest that FLT3 and SUCLG1 play a role in the same pathway as POLRMT, regulating leukemia cell proliferation.

[0180] Next, the development of leukemia was tested in vivo. FLT3 ITD alone was unable to cause leukemia formation, so FLT3 ITD was introduced into a MLL-AF9-driven leukemia model (Stubbs et al, 2008). HA-tagged FLT3 ITD was co-transduced with MLL-AF9 into mouse bone marrow cells to establish a mouse leukemia model Figure 5 Mid F). Further, shRNA targeting Suclg1 was stably expressed in leukemia bone marrow cells, and the stably transduced bone marrow cells were transplanted into sublethally irradiated recipients Figure 5Middle G). Knockdown of Suclg1 significantly prolonged the survival time of animals ( Figure 5 Middle H). Thus, SUCLG1 and POLRMT are essential for the development of FLT3 mutant-driven leukemia.

[0181] Next, the stably transfected cell line MV411 was transplanted into sublethally irradiated NSG mice to establish a humanized leukemia model. WT Knockdown of SUCLG1 in MV411 cells significantly delayed the development of leukemia, whereas in the presence of POLRMT K622R A weak inhibitory effect was observed in the leukemia model established with the mutant ( Figure 5 These data suggest that mutant FLT3 promotes leukemia progression by regulating succinylation of POLRMT.

[0182] To determine the pathological relevance of POLRMT succinylation, human normal and leukemic bone marrow samples were collected and sequenced to determine the FLT3 gene mutation status. WT AML n = 5; FLT3 ITD / TKD AML n = 10), the protein expression level in whole cell lysates and the K622 succinylation level of immunopurified POLRMT were detected. Metabolite quantitative analysis showed that both FLT3 WT Still FLT3 ITD / TKD In AML samples, the levels of succinyl-CoA were lower than those in healthy controls ( Figure 5 In FLT3-mutated AML samples, K622 succinylation was significantly decreased, while POLRMT protein expression did not change significantly ( Figure 5 Consistent with this result, SUCLG1 protein was overexpressed in FLT3 mutant AML samples ( Figure 5 The relationship between SUCLG1 and leukemia development was further explored in the TCGA AML dataset. The mRNA level of SUCLG1 was significantly increased in patients carrying FLT3 mutants, while the mRNA level of POLRMT did not change significantly ( Figure 5 MN). It is worth noting that in FLT3 WT In patients with FLT3, high SUCLG1 expression predicted worse overall survival, but TKD / ITD There was no significant association among individuals ( Figure 5 This clinical evidence suggests that FLT3 and SUCLG1 may have other functions in regulating mitochondrial biogenesis and promoting the occurrence and development of leukemia.

[0183] In summary, SUCLG1 limits the succinylation of POLRMT by inhibiting the level of succinyl-CoA and enhances mitochondrial activity. Mutant FLT3 upregulates SUCLG1 to remodel mitochondrial biogenesis and promote AML progression Figure 5 P) in the middle.

[0184] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the original description and drawings are to be construed as merely illustrative of preferred embodiments of the application and not restrictive thereof.

Claims

1. Use of an inhibitor targeting SUCLG1 for preparing a composition or formulation for: (1) Promote the succinylation modification of mitochondrial RNA polymerase (POLRMT), inhibit POLRMT activity, and thus reduce mitochondrial biogenesis; and / or (2) Treating or preventing mitochondrial biogenesis-dependent tumors or cancers.

2. The use according to claim 1, characterized in that The inhibitor targeting SUCLG1 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 2, characterized in that The inhibitor targeting SUCLG1 is shRNA, 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.

4. The use according to any one of claims 1 to 3, characterized in that The inhibitor targeting SUCLG1 inhibits POLRMT activity by promoting succinylation modification of the 622th lysine site (K622) of POLRMT; The "inhibiting POLRMT activity" includes: disrupting the binding of POLRMT with mitochondrial transcription factors TFAM and TFB2M; disrupting the binding of POLRMT with mtDNA; and inhibiting mitochondrial gene transcription expression.

5. The use according to any one of claims 1 to 3, characterized in that The mitochondrial biogenesis-dependent tumors or cancers include (but are not limited to) acute myeloid leukemia, colorectal cancer, liver cancer, lung cancer, etc.

6. The use according to claim 5, characterized in that The mitochondrial biogenesis-dependent tumor or cancer is acute myeloid leukemia, preferably FLT3 mutant acute myeloid leukemia.

7. An active ingredient combination, characterized in that The active ingredient combination includes: (i) a first active ingredient, wherein the first active ingredient is an inhibitor targeting SUCLG1; Preferably, the active ingredient combination further comprises: (ii) a second active ingredient, wherein the second active ingredient is an inhibitor targeting POLRMT.

8. Use of a combination of active ingredients for preparing a composition or formulation for: reducing mitochondrial biogenesis; and / or treating or preventing mitochondrial biogenesis-dependent tumors or cancers; The active ingredient combination comprises a first active ingredient that targets an inhibitor of SUCLG1, and a second active ingredient that targets an inhibitor of POLRMT.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) an inhibitor targeting SUCLG1, (b) an inhibitor targeting POLRMT, and (c) a pharmaceutically acceptable carrier.

10. A medicine box, characterized in that: The medicine box comprises: (C1) a first formulation in a first container, the first formulation comprising an inhibitor targeting SUCLG1 as a first active ingredient, and a pharmaceutically acceptable carrier; (C2) a second formulation in a second container, the second formulation comprising an inhibitor targeting POLRMT as a second active ingredient; and a pharmaceutically acceptable carrier; and (C3) instructions, wherein the instructions indicate that the kit is used to treat and / or prevent tumors or cancers.

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