A polypeptide capable of specifically inhibiting mre11 k673 lactoylation and uses thereof

Abstract

The present application provides an isolated polypeptide, any one selected from the polypeptide containing the amino acid sequence as shown in SEQ ID NO: 3; or the fragment containing at least SSTSSSKIMSQSQ in the amino acid sequence shown in SEQ ID NO: 3. The present application also provides an isolated polynucleotide, the sequence of which is selected from: the coding sequence of the above-mentioned polypeptide; or the complementary sequence of the above-mentioned coding sequence. The present application also provides the use of the above-mentioned polypeptide in the preparation of a drug for specifically inhibiting Mre11K673 lactylation. The present application also provides the use of the above-mentioned polypeptide in the preparation of a drug for promoting the sensitivity of cancer cells to chemotherapy. The present application also provides the use of the lactylation-modified Mre11K673 in the screening of a drug for treating cancer, which is a drug for specifically inhibiting Mre11K673 lactylation. The combination of the polypeptide of the present application and the chemotherapeutic drug significantly promotes the chemotherapy-mediated cancer cell killing effect in vitro and in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a polypeptide and its uses, specifically a polypeptide that can specifically inhibit Mre11 K673 lactation and its uses. Background Technology

[0002] The Warburg effect, also known as aerobic glycolysis, is a metabolic hallmark of most cancers, referring to the production of large amounts of lactate even in the presence of oxygen in cancer cells. This intermediate metabolite, lactate, is produced from pyruvate catalyzed by lactate dehydrogenase (LDH) in cancer cells and has been reported to play crucial roles in various cellular processes, including promoting tumor cell proliferation as an energy source, tumor cell metastasis, and immunosuppression. However, its non-metabolic functions in cancer cells remain largely unknown. Recently, Professor Yingming Zhao's team at the University of Chicago discovered that lactate is a major source of histone lactation. In addition to its metabolic functions, lactate also induces a previously unknown post-translational modification (PTM) called lactation. They found that lactate induces lactation of histone lysine residues, thereby regulating gene transcription and expression. Studies have shown that lactate can promote chemotherapy resistance. However, the exact mechanism of this phenotype remains to be explored.

[0003] Maintaining genome integrity is crucial for ensuring faithful replication and reliable transmission of genetic material. DNA damage and replication errors caused by endogenous and exogenous factors are major contributors to genomic instability. Among these, DNA double-strand breaks (DSBs) are the most damaging form of DNA damage, posing a significant threat to genomic instability. Defects in DSB repair are involved in a variety of diseases, including aging, cancer, neurological disorders, growth retardation, and immune deficiencies.

[0004] In current models, two main pathways are responsible for repairing DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR). Compared to HR, NHEJ is much faster but error-prone, as it directly joins the broken ends together, potentially introducing numerous mutations. Unlike NHEJ (which occurs throughout the cell cycle), HR occurs strictly during the S and G2 phases because it requires a homologous template. Therefore, HR is the error-free mechanism for DSB repair. One of the key steps in HR is end excision of damaged DNA to produce suitable single-stranded DNA. In mammals, end excision is initiated by the Mre11 / RAD50 / NBS1 (MRN) complex and then promoted by phosphorylated CtIP (CTBP-interacting protein, a DSB-activated DNA endonuclease). The MRN complex is a sensor and multifunctional complex for DSBs, playing a crucial role in the sensing, processing, and repair of DNA double-strand breaks.

[0005] HR (human retrieval of DNA) is one of the most important types of DNA damage repair, error-free, and plays a crucial role in maintaining genomic stability and suppressing tumorigenesis. On the other hand, high activation of HR in cancer is associated with chemotherapy resistance. Mre11, a core component of the MRN complex, is responsible for DNA binding and cleavage. After DSBs (disruptive stem cell block syndromes), Mre11 is recruited to the damage site to initiate DNA end excision. Subsequently, the exonuclease EXO1 / DNA 2 is further recruited to the DNA damage site, mediating extensive excision, a key process in HR repair. Therefore, the recruitment of Mre11 to DNA is a critical step in Mre11-mediated DNA excision and the initiation of HR repair.

[0006] Dysregulation of Mre11 leads to increased susceptibility to immunodeficiency, radiation sensitivity, cell cycle checkpoint defects, and cancer development. Therefore, precise regulation of Mre11 is crucial for protecting genome stability and cell viability. Post-translational modifications of Mre11 play a significant role in regulating its function. UBQLN4-mediated Mre11 ubiquitination leads to its degradation, thereby affecting the selection of DSB repair pathways. UFL1-mediated UFMylation of Mre11 on K282 is essential for optimal ATM activation, homologous recombination-mediated DSB repair, and genome integrity. PLK1-mediated phosphorylation of Mre11 reduces its DNA-binding ability, thereby inhibiting HR repair. PRMT1 methylation of arginine in its GAR domain regulates its exonuclease activity. Furthermore, C1QBP promotes Mre11 stability but inhibits its exonuclease activity by interacting with Mre11 to prevent its DNA binding. DYLL1 and Nej1 (yeast homologs of XLF) have also been reported to bind directly to Mre11 and then inhibit Mre11 excision. Current models suggest that Mre11 binds to DNA in a RAD50-dependent and independent manner. However, the regulation and mechanisms of Mre11 recruitment to DNA in response to DNA damage remain largely unknown.

[0007] Mre11 is a core component of the MRN complex, possessing both endonuclease and 3'-5' exonuclease activities. Mre11 contains two DNA-binding domains (DBDs), enabling it to bind to either single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Upon a DNA double-strand break, Mre11 is immediately recruited to DSB sites and cleaves the damaged DNA by initiating end-cutting in cooperation with CtIP. Subsequently, long-distance DNA excision is completed via exonucleases EXO1 and DNA2. Simultaneously, the resulting ssDNA is rapidly wrapped and protected by replication protein A (RPA) and then replaced by RAD51 recombinase, which is crucial for finding homologous templates and subsequent recombinant regeneration (HR) processes. A growing body of research suggests that overactivation of HR is a major cause of chemotherapy resistance in cancer. Metabolic abnormalities are one of the most prominent features of cancer cells. The most well-known metabolic alteration in cancer cells is the Weinberg effect, defined as a significant increase in glycolysis even under oxygen-rich conditions. However, it remains unclear whether HR is influenced by the Weinberg effect. Summary of the Invention

[0008] The purpose of this invention is to provide a cell-penetrating peptide that can specifically inhibit Mre11 K673 lactation and its uses. This peptide that can specifically inhibit Mre11 K673 lactation and its uses are intended to solve the technical problem that existing drugs have poor therapeutic effects on cancers caused by metabolic abnormalities.

[0009] This invention provides an isolated polypeptide, any polypeptide selected from the following:

[0010] (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO:3;

[0011] (2) A fragment containing at least SSTSSSKIMSQSQ in the amino acid sequence shown in SEQ ID NO:3.

[0012] Preferably, the amino acid sequence of the isolated polypeptide is shown in SEQ ID NO.3 (HLYVSPWGGSSTSSSKIMSQSQ).

[0013] The present invention also provides an isolated polynucleotide whose sequence is selected from:

[0014] (1) The coding sequence of the isolated polypeptide; or

[0015] (2)(1) The complementary sequence of the encoded sequence.

[0016] The present invention also provides an expression vector containing the above-mentioned polynucleotides.

[0017] The term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host cell. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements. They can be used to transform appropriate host cells to enable them to express polypeptides.

[0018] The present invention also provides a pharmaceutical composition comprising the above-described polypeptide and a pharmaceutically or physiologically acceptable carrier.

[0019] This invention also provides the use of the above-isolated polypeptides, isolated polynucleotides, and expression vectors in the preparation of drugs for specifically inhibiting Mre11 K673 lactation. This invention further provides the use of the above-isolated polypeptides, isolated polynucleotides, and expression vectors in any of the following:

[0020] 1) To prepare drugs that improve the sensitivity of cancer cells to chemotherapy drugs;

[0021] 2) Prepare drugs that enhance the therapeutic effect of chemotherapy drugs.

[0022] Furthermore, the chemotherapy drug is one that can induce double-strand breaks in DNA.

[0023] Furthermore, the chemotherapy drugs mentioned are platinum-based or PARP inhibitor chemotherapy drugs.

[0024] Furthermore, the cancer cells mentioned are colorectal cancer cells, breast cancer cells, or ovarian cancer cells.

[0025] This invention also provides the use of lactated Mre11 K673 in screening drugs for treating cancer, the screening steps being as follows: Figure 5 A-5B.

[0026] The present invention also provides the use of the first component together with the second component in the preparation of a medicament for treating cancer, wherein the first component is composed of the above-described pharmaceutical composition, and wherein the second component is composed of a chemotherapy drug.

[0027] Furthermore, the chemotherapy drug is one that can induce double-strand breaks in DNA.

[0028] Furthermore, the chemotherapy drugs are platinum-based or PARP inhibitor chemotherapy drugs. Furthermore, the cancer is colorectal cancer, breast cancer, or ovarian cancer.

[0029] In this invention, Mre11 is lactated by CBP (CREB-binding protein, an acetyltransferase). Following DNA damage, the interaction between Mre11 and CBP increases, leading to increased Mre11 lactation. Furthermore, Mre11 is lactated at its second DNA-binding domain, K673. Interestingly, our findings indicate that Mre11 lactation enhances its DNA-binding capacity, subsequently promoting end-cutting and heart rate (HR) repair. High Mre11 lactation results in highly efficient HR repair and chemoresistant effects. Therefore, combining a CBP inhibitor (CBPi) or an LDH inhibitor (LDHi) that inhibits Mre11 lactation with chemotherapy can significantly attenuate HR repair and increase cancer-killing efficacy. Furthermore, we conducted in vivo cancer-killing assays using a highly lactated colon cancer PDX model, demonstrating that inhibiting Mre11 K673 lactation significantly enhances PARPi-mediated colon cancer killing.

[0030] Lactic acidification is a novel post-translational modification induced by lactate or sodium lactate. The Mre11 protein plays a crucial role in homologous recombination repair (HR) and maintaining genomic stability. Our invention reveals that Mre11 undergoes lactation at position 673 after DNA damage, a process catalyzed by CBP acetyltransferase. Lactic acidification of Mre11 promotes its binding to DNA, thereby facilitating DNA end-cutting and homologous recombination repair (HR). Inhibition of CBP or LDH downregulates Mre11 lactation, leading to HR inhibition and enhancing the sensitivity of a colorectal cancer patient-derived xenograft (PDX) model to chemotherapy.

[0031] This invention demonstrates that lactation regulates heart rate (HR). Mechanistically, Mre11 is lactated following DNA damage. CBP catalyzes lactation modification of Mre11 at K673, located in its second DNA-binding domain (DBD). Mre11 lactation plays a crucial role in regulating Mre11 binding to DNA and DNA end excision. High Mre11 lactation promotes HR and chemotherapy resistance in cancer cells.

[0032] This invention reveals that lactated Mre11 is a novel regulatory protein of heart rate repair (HR) that links cancer metabolism with DSB repair. Given the Weinberg effect, which generates large amounts of lactate in most tumors, protein lactation may enhance HR repair in cancer and lead to chemotherapy resistance. Simultaneously, DNA damage also enhances Mre11 lactation by activating CBP. Internal factors (lactate generated by the Weinberg effect) and external factors (chemotherapy-induced hyperlactatosis of Mre11) may overactivate HR during tumor development and treatment, leading to chemotherapy resistance.

[0033] Compared with existing technologies, the technological advancements of this invention are significant. Our findings indicate that Mre11 lactation promotes heart rate (HR), and targeting Mre11 lactation may be an effective strategy to overcome chemotherapy resistance in cancer. This invention designs and screens a cell-penetrating peptide (CPP) that specifically inhibits Mre11 lactation, which inhibits DNA end-cutting and DNA damage repair. This peptide significantly inhibits Mre11 K673 lactation and suppresses HR, thereby increasing the sensitivity of cancer cells to chemotherapy. The peptide of this invention significantly inhibits HR, thereby promoting the sensitivity of cancer cells to cisplatin and PARP inhibitors (PARPi). The combination of this peptide and chemotherapeutic drugs (cisplatin or olaparib) significantly promotes chemotherapy-mediated cancer cell killing in vitro and in vivo. Targeting Mre11 lactation has potential therapeutic strategies. This invention also discovers that key proteins in the HR pathway can undergo lactation modification and link cancer metabolism with DSB repair. Attached Figure Description

[0034] Figure 1 This demonstrates that CBP-mediated lactation modification of Mre11 K673 is achieved.

[0035] Figure 2 The results showed that Mre11 lactation modification promotes its binding to DNA and recruitment to DNA damage sites.

[0036] Figure 3 The results showed that Mre11 lactation modification promotes DNA end cleavage and homologous recombination repair.

[0037] Figure 4 High Mre11 lactation modification was shown to promote chemotherapy tolerance.

[0038] Figure 5 The study demonstrated that targeting Mre11 lactation modification with short peptides in combination with chemotherapeutic drugs significantly improved the tumor-killing effect. Detailed Implementation

[0039] The experimental materials and procedures of this invention are described below:

[0040] Cell culture, plasmids, reagents and antibodies

[0041] HEK293T, U2OS, ovarian cancer cell line OV-car8, breast cancer cell line MBA-MD-231, and colon cancer cell lines HCT116 and RKO were purchased from ATCC. Unless otherwise specified, cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% (v / v) CO2. RKO cells were cultured in RPMI1640 supplemented with 10% FBS. Mre11 WT and Mre11K673R were subcloned into plvx3 and PEGX-4T-2 vectors (Clontech). Mre11K510R, K609R, K625R, and K673R mutants were constructed using a two-step mutagenesis method.

[0042] Sodium L-lactic acid (71718), LDHi (O2751), puromycin, cisplatin (P4394), 5-bromo-2′-deoxyuridine (BRDU), ATM inhibitor KU55933, and etoposide (E1383) were purchased from Sigma-Aldrich. CBP inhibitor (SGC-CBP30) was purchased from Selleckchem. Olaparib and MCT1 / 4 inhibitor (Syrosingopine, Su-3118) were purchased from Selleckchem.

[0043] The following antibodies were used in this invention: anti-GAPDH (Proteintech, 60004-1-lg), anti-β-actin (Proteintech, 66009-1-Ig), anti-γH2AX (EMD Millipore: 2884537, 1:1000), anti-Flag (Sigma: F1804, 1:2000), anti-HA (Cell Signaling Technology: no. 3724, 1:2000), anti-RPA32 (Santacruz: sc-56770, 1:1000), anti-RAD51 (GENETEX: GTX100469, 1:1000), anti-BRDU (BD: 347580, 1:100), anti-Mre11 (GENETEX: GTX70212, 1:1000), and anti-RAD50 (GENETEX: GTX70228). The anti-NBS1 (Novus:NB100-143, 1:1000), anti-pan-Kla (PTM Biolab:PTM-1401RM, 1:1000), anti-LDHA (CellSignaling Technology:no.2012S, 1:1000), anti-LDHB (Millipore Sigma:ABC927, 1:1000), anti-H3 (Proteintech,no.17168-1-AP, 1:2000) and anti-Mre11-K673la (1:1000) are produced by PTM Biolab.

[0044] DNA transfection, viral packaging, and lentiviral infection

[0045] Cells were transfected with polyetherimide (PEI) or Lipofectamine 2000 (Thermo Fisher Scientific). Viral supernatant was collected twice (24 hours and 48 hours) after co-transfection with lentiviral vector and packaging plasmids (pMD2G and pSPAX2). The harvested lentivirus was added to the cells, and further experiments were performed with 8 μg / mL polyamine to improve infection efficiency. Stable cells were selected using medium containing 2 μg / mL puromycin. Stable cells were detected by Western blotting.

[0046] HR report analysis

[0047] DSB repair efficiency was examined using the DR-GFP and EJ5-GFP reporter systems. Briefly, HEK293T cells stably expressing the described shRNA or normal HEK293T cells were seeded into six-well plates in triplicate. After 24 hours, cells in each well were transfected with a complex containing 3 μL polyetherimide (PEI), 500 ng DR-GFP (or 500 ng EJ5-GFP), 100 ng mCherry, and 500 ng I-SceI plasmid. 24 hours post-transfection, cells were treated with the specified reagents. 48 hours post-transfection, cells were collected and analyzed by flow cytometry (FACS), sorted by RFP-positive cells or RFP / GFP-positive cells. Repair efficiency ratios were calculated as described above.

[0048] Immunofluorescence

[0049] 24 hours prior to treatment, cells were seeded into six-well plates with coverslips. For RRA32 lesion staining, cells were fixed for 20 minutes at -20°C with methanol:acetone (1:1). Cells were washed twice with PBS and then refixed for 15 minutes at room temperature with 4% PFA. For RAD51 staining, cells were pre-extracted on ice for 10 minutes with pre-extraction buffer (20 mM HEPES, pH 7.4, 20 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT and protease inhibitor mixture) and then fixed for 10 minutes with 4% PFA. Before staining, cells were blocked with 2% goat serum for 1 hour at room temperature. Cells were then incubated overnight at 4°C with primary antibody. After washing three times with PBS, cells were incubated with secondary antibody at room temperature for 1 hour. After washing three times with PBS, cells were incubated with DAPI at room temperature for approximately 2 minutes. After washing, cells were fixed with anti-fading solution and observed using a Nikoneclipse80i fluorescence microscope.

[0050] BRDU staining for end excision detection

[0051] U2OS cells seeded on coverslips were pre-incubated with 20 μg / ml BRDU for 24 h, followed by treatment with cisplatin for 12 h. Cells were washed with PBS and then released on ice for 10 min with buffer 1 (10 mM PIPE, pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100). Next, cells were washed with PBS and released again on ice with buffer 2 (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 3 mM MgCl2, 1% Tween-40, 0.5% sodium deoxycholate) for 10 min. After washing, cells were fixed with 4% PFA for 30 min at room temperature. Slides were blocked with 5% BSA and then incubated overnight at 4 °C with anti-BRDU (1:100) primary antibody. Secondary antibody and DAPI staining were performed as described above.

[0052] Mass spectrometry analysis

[0053] To identify the Mre11 lactation site, 293T cells stably expressing the HA-labeled Mre11 were treated with 50 mM NALA for 24 h prior to harvest, followed by lysis. The lysates were purified using anti-HA agarose beads. The particles were then analyzed on SDS-PAGE and stained with Coomassie blue. The bands corresponding to HA-Mre11 were excised and sent for mass spectrometry analysis.

[0054] Immunoprecipitation and Western blotting

[0055] For transient transfection and co-immunoprecipitation assays, HEK293T cells were transiently co-transfected with constructs encoding HA-labeled Mre11 or HA-labeled CBP constructs. Transfected cells were lysed on ice for 25 min using NETN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) containing 1× protease inhibitor. After removing cell debris by centrifugation at 12,000 rpm for 10 min, the soluble fraction was collected and incubated with HA beads at 4°C for 2 h. The HA beads were washed three times with NETN buffer, boiled in 1× SDS loading buffer for 5 min, and analyzed on SDS-PAGE. The membrane was blocked with 5% milk in TBST buffer and then probed with antibody as instructed.

[0056] HRD Score Analysis

[0057] The HRD score for the TCGA breast cancer cohort was published earlier and is available for download from the Genomic Data Sharing (GDC). Log data for TPM expression conversion was downloaded from the UCSCxenabrowser. HRD scoring analysis was performed on the basal subtypes (n=140) based on previously defined PAM50 features. Samples were grouped according to median expression levels of BRCA1 and LDHA. Statistical significance was tested using the Mann-Whitney-Wilcoxon test.

[0058] Whole cell extract and chromatin component extract

[0059] First, cells were lysed on ice for 10 min on ice using NETN buffer containing 1× protease inhibitor (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40). After centrifugation (12000 rpm, 4°C for 10 min), the supernatant was collected as the whole-cell extract. The remainder was washed at least three times with PBS buffer and further lysed with cold EBC2 buffer (50 mM Tris HCl [pH 7.5], 300 mM NaCl, 5 mM CaCl2, and 10 U micronuclease). After sonication and centrifugation (12000 rpm, 4°C for 10 min), the supernatant was collected as the chromatin fraction.

[0060] Colony formation assay

[0061] Indicated cells (500-2000) were seeded in triplicate into each well of a six-well plate. For chemosensitivity assays, cells were exposed to the specified dose of the specified drug one day later and incubated for 10-14 days to allow colony formation. Colons were stained with GIEMSA and quantified. Results were normalized to electroplating efficiency.

[0062] Electrophoretic mobility transfer assay

[0063] The specified concentration of Mre11 protein was incubated with dsDNA or pendant DNA substrate at 37°C for 15 minutes in a reaction buffer containing 100 mM KCl (25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 100 mg / ml BSA, 2 mM ATP). After incubation, the reaction mixture was mixed with a loading buffer (20 mM Tris-HCl, pH 7.5, 40% glycerol, 2 mM EDTA, 0.2% Orange G) and then electrophoresed on a 0.5% agarose gel in SB buffer (10 mM NaOH, 40 mM boric acid, pH 8.0).

[0064] Peptide synthesis

[0065] All peptides were synthesized by Guoping Pharmaceutical Co., Ltd. (China Hehui). The synthesized peptides were purified in vitro and in vivo by high-performance liquid chromatography to a purity >98%. The amino acid sequence of the peptides used in vivo was of the D isotype. For in vitro experiments, the peptides were dissolved in PBS to prepare a 10 mM stock solution. For in vivo use, K673-pe and K673R-pe were dissolved in PBS and kept on ice until injection. The solution was brought to room temperature before injection.

[0066] organoid models derived from colorectal cancer patients

[0067] For the first time, pantothenate levels in four colon cancer organoids were determined using Western blotting. Furthermore, based on their pantothenate levels, these organoids were divided into two groups: high-lactate and low-lactate. Organoids in good condition were passaged and seeded in standard 96-well cell culture plates (Corning). Organoids with high lactate levels were treated with cisplatin alone or in combination with LDHi or CBPi. Organoids with low lactate levels were treated with cisplatin alone or in combination with lactate or sodium lactate. Organoid growth was assessed using CCK8 assay after 72 hours.

[0068] animal

[0069] All animal experiments and procedures were conducted in strict accordance with the "Guidelines for the Care and Use of Laboratory Animals" developed by the National Institutes of Health (NIH) (National Academy of Sciences Press, 2011) and in accordance with the ethical guidelines approved by Tongji University School of Medicine.

[0070] PDX model for colorectal cancer patients

[0071] Xenografts (PDX) derived from colorectal cancer patients were subcutaneously transplanted into 6-week-old female nu / nu mice. Mice carrying 150-200 mm... 3 Mice with tumors were randomly assigned to six groups: a solvent control group (saline), an oxalate group (1000 mg / kg), a CBPi group (15 mg / kg), an olaparib group (50 mg / kg), an olaparib (50 mg / kg) + oxalate (1000 mg / kg), and an olaparib (50 mg / kg) + CBPi (15 mg / kg). Mice were treated with intraperitoneal injections three times a week. Tumor volume was measured and assessed as described in the Tumor Xenograft Assay.

[0072] For the assay of killing colon cancer PDX in combination with K673-pe or K673R-pe and olaparib, mice carrying tumors of 150-200 mm3 were randomly divided into six groups as described above: solvent control group (saline), K673R-pe (5 mg / kg), K673-pe (5 mg / kg), olaparib (50 mg / kg), olaparib (50 mg / kg) + K673R-pe (5 mg / kg, 5 mg / kg), and olaparib (50 mg / kg) + K673-pe (5 mg / kg). K673-pe or K673R-pe was administered intraperitoneally once daily, and olaparib was administered intraperitoneally three times weekly.

[0073] Quantitative and statistical analysis

[0074] For cell survival, PDO, PDX xenograft, and lesion assays of RPA2, Mre11, RDA51, BRDU, and Mre11-k673la, data are presented as mean ± SEM of three independent experiments. For animal assays, data are presented as mean ± SEM of five mice. Statistical analysis was performed using the Student t-test or χ² test in GraphPad Prism.

[0075] Example 1: Mre11 can undergo lactation modification.

[0076] Given that lactation is a novel post-translational modification, its role in cells remains largely unknown. Since LDHA is the main kinase catalyzing lactate production, it is possible that LDHA regulates protein lactation modifications. Interestingly, we found that patients with low LDHA expression exhibited higher HRD scores, indicating a deficiency in HR. Furthermore, treatment of cells with sodium lactate (NALA) significantly increased cellular pan-lactation levels and the efficiency of homologous recombination repair (HR), suggesting that high levels of protein lactation modifications may play a crucial role in HR repair. Figure 1 A-1C).

[0077] By screening several key HR proteins, we found that Mre11 undergoes strong lactation modification in cells. Figure 1 D). Sodium lactate treatment enhanced the lactation of Mre11, while LDHi treatment had the opposite effect. Figure 1 E). Knockdown of LDHA / B significantly reduced Mre11 lactation ( Figure 2 C). After screening multiple acetyltransferases, we found that CBP is the main acetyltransferase mediating the lactation of Mre11. Figure 2 F). Inhibition or knockdown of CBP significantly reduces Mre11 lactation ( Figure 1 G).

[0078] Mre11 is an evolutionarily conserved protein, primarily composed of a nuclease domain, two DBD domains, and a GAR domain. Figure 1 H). To identify the lactation site of Mre11, we used purified HA-labeled Mre11 protein for mass spectrometry (MS) analysis. Figure 2 J, K673 was identified as a potential Mre11 lactation site. Further site mutation experiments revealed that K673, located within the second DBD domain, is the primary site for Mre11 lactation modification in response to DNA damage. Figure 1 K).

[0079] Example 2: Mre11 lactation modification promotes its binding to DNA and recruitment to DNA damage sites.

[0080] Because Mre11 possesses a DNA-binding domain, its binding to DNA has been reported to occur in a RAD50-dependent or RAD50-independent manner. However, the mechanism of Mre11 binding to DNA remains not fully understood. To investigate whether lactation affects the binding of Mre11 to DNA, we first performed in vitro lactation on purified Mre11 WT, followed by in vitro DNA binding experiments. Figure 2 As shown in Figure A, electrophoretic mobility transfer assay (EMSA) results indicate that lactation promotes the binding of Mre11 to dsDNA and pendant DNA. Furthermore, compared to the Mre11 KR mutant, WT Mre11 exhibits a higher affinity for DNA binding. Figure 2 B). Furthermore, sodium lactate (NALA) treatment enhanced the formation of Mre11 or Mre11-K673la foci and the recruitment of Mre11 to chromatin (B). Figure 2 C-2D). Simultaneously, by inhibiting CBP or LDH, the reduction in Mre11 chromatin recruitment was significantly reduced (C-2D). Figure 2 E-2F). Furthermore, compared to Mre11 WT, Mre11 KR recruited significantly less chromatin (E-2F). Figure 2 E-2F). Similarly, Mre11 KR showed less focal formation after DNA damage ( Figure 2 In summary, our findings suggest that lactation of Mre11 is important for its binding to DNA and for the formation of DNA damage-induced focal points.

[0081] Example 3: Mre11 lactation modification promotes DNA end cleavage and homologous recombination repair.

[0082] To further determine whether lactation affects DNA end excision, we first treated cells with sodium lactate or LDHi, then examined p-RPA2 levels and BRDU focal formation. Inhibition of LDHA / B significantly reduced cisplatin-induced p-RPA2 levels (…). Figure 3 A). Sodium lactate treatment significantly increased the formation of DNA damage-induced BRDU foci ( Figure 3 B). Furthermore, sodium lactate can salvage the weakening of p-RPA2 caused by the inhibition of LDHA / B (B). Figure 3 A).

[0083] To further investigate the role of Mre11 K673 lactation in DNA end-removal, we constructed tumor cells stably expressing Mre11 WT and Mre11 KR (with endogenous Mre11 knocked down). We found that Mre11 KR cells suppressed DNA damage-induced p-RPA2 levels ( Figure 3 C). Furthermore, sodium lactate treatment significantly enhanced the level of DNA damage p-RPA2 in Mre11 WT cells, but this phenomenon was not observed in Mre11 KR cells. Figure 3 C). Furthermore, the Mre11 KR mutation impairs the formation of DNA damage-induced RPA2 and RAD51 focal points (C). Figure 3 (D-3E). Since Mre11 lactation enhances DNA excision, a key initiation step in HR repair, we next tested whether Mre11 lactation modulates HR repair and genome stability. Figure 3 As shown in Figure F, the Mre11 KR mutation reduces HR repair. Furthermore, by examining γH2AX levels after DNA damage, we found that the Mre11 KR mutation impairs DNA damage repair. Figure 3 G).

[0084] Example 4: High Mre11 lactation modification promotes chemotherapy tolerance

[0085] Because Mre11 lactation enhances DNA excision and promotes heart rate repair (HR) repair, highly activated HR repair leads to tumor cell resistance to PARP inhibitors or platinum-based chemotherapy drugs. Next, we will examine whether Mre11 lactation modification affects the killing effect of chemotherapy drugs on tumor cells. Figure 4 As shown in A-4B, sodium lactate treatment led to greater resistance of cancer cells to olaparib, while combined CBPi treatment reversed this effect. Furthermore, Mre11 KR cells showed greater sensitivity to olaparib treatment compared to Mre11 WT cells. Figure 4C-4D). Simultaneously, manipulation of lactation via CBPi or NALA affected the chemotherapy response of Mre11WT cells, but had no effect on Mre11 KR mutant cells. Figure 4 These results preliminarily suggest that Mre11 lactation regulates the response of tumor cells to chemotherapy in a cell-based model.

[0086] To further confirm the role of Mre11 lactation in chemotherapy, we used highly Mre11 lactated PDX in vivo for tumor killing experiments. Figure 4 As shown in E-4G, both CBPi and LDHi treatments significantly enhanced the killing effect of olaparib on high K673 lactic acidified colonic PDX. These results suggest that CBPi or LDHi combined with chemotherapy may be a potentially effective strategy for the treatment of Mre11 K673 high lactic acidified cancer.

[0087] Example 5: Short peptide-specific targeting of Mre11 lactation modification combined with chemotherapy drugs significantly improved the tumor-killing effect.

[0088] Given that targeting CBP or LDH may affect multiple signaling pathways, a strategy specifically targeting Mre11K673 lactation is needed. Based on the motifs of protein post-translational modifications (PTMs), specific peptide generation has proven to be a promising and effective strategy for inhibiting PTMs.

[0089] After analyzing the sequence near the Mre11 K673 site, this invention, combining previous literature reports and based on the lactation-modified sequence of Mre11, designed a short peptide of approximately 13 amino acids in length, including the Mre11 K673 site, plus a cell-penetrating peptide. This peptide is fused with five cell-penetrating peptides (CPPs) (which have become one of the most commonly used and efficient tools for intracellular delivery of biomolecules). Figure 5 A).

[0090] Specifically, the sequence of the polypeptide is as follows:

[0091] K673-Peptide-1#:HLYVSPWGGTSSSKIMSQSQVS(SEQ ID NO.1);

[0092] K673-Peptide-2#:HLYVSPWGGRWSSTSSSKIMSQ(SEQ ID NO.2);

[0093] K673-Peptide-3#:HLYVSPWGGSSTSSKIMSQSQ(SEQ ID NO.3);

[0094] K673-Peptide-4#:HLYVSPWGGDQRWSSTSSSKIM(SEQ ID NO.4);

[0095] K673-Peptide-5#:HLYVSPWGGSSKIMSQSQVSKG (SEQ ID NO. 5).

[0096] K673-peptide-3# (referred to as K673-pe) exhibits the strongest inhibitory effect on the lactation of Mre11. Figure 5 B). Based on the K673-pe peptide, we synthesized a control peptide (called K673R-pe), in which the K673 residues were replaced by R residues. Figure 5 As shown in C-5E, K673-pe significantly inhibited the formation of DNA damage-induced Mre11-k673la and RPA2 foci, indicating that K673-pe inhibited Mre11 K673 lactation, leading to a significant reduction in DNA end excision. Furthermore, K673-pe treatment also reduced the formation of DNA damage-induced RAD51 foci, consistent with the significant decrease in HR levels after K673-pe treatment. Figure 5 These results indicate that the K673-pe peptide can downregulate HR by inhibiting Mre11 lactation.

[0097] Next, we will investigate whether K673-pe promotes the sensitivity of cancer cells to chemotherapy drugs. For example... Figure 5 As shown in H-5K, K673-pe, rather than K673R-pe, significantly enhanced the colorectal cancer cells HCT11-6 (…). Figure 5 H), colorectal cancer cells RKO ( Figure 5 I) Ovarian cancer cells ( Figure 5 J), breast cancer cells ( Figure 5 K) is sensitive to cisplatin or PARP inhibitors.

[0098] To further confirm the synergistic killing effect of K673-pe combined with chemotherapy in vivo, we conducted tumor killing experiments using a highly lactated Mre11K673 colon cancer PDX model. Figure 5 As shown in L-5M, K673-pe, rather than K673R-pe, significantly enhanced the sensitivity of colonic PDX to olaparib. These results suggest that K673-pe targeting Mre11 K673 lactation modification is a potential strategy to enhance the efficacy of chemotherapy in tumors with high Mre11 K673 lactation modification.

Claims

1. An isolated polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3.

2. An isolated polynucleotide, characterized in that, The sequence of the polynucleotide is selected from: (1) The coding sequence of the polypeptide according to claim 1; or (2) The complementary sequence of the coding sequence of the polypeptide of claim 1.

3. An expression vector containing the polynucleotide of claim 2.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: 1) The polypeptide of claim 1; and 2) A pharmaceutically or physiologically acceptable carrier.

5. Use of the polypeptide of claim 1, the polynucleotide of claim 2, and the expression vector of claim 3 in the preparation of a medicament for treating colorectal cancer, breast cancer, or ovarian cancer.

6. The use of the first component together with the second component in the preparation of a medicament for treating colorectal cancer, breast cancer, or ovarian cancer, wherein the first component is composed of the pharmaceutical composition of claim 4, and the second component is cisplatin or olaparib.

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