Lactylation modified XLF protein, preparation for inhibiting XLF lactylation and application

Abstract

The invention relates to a lactylation modified XLF protein, a preparation for inhibiting XLF lactylation and application. The research finds that the function of the XLF in the NHEJ process is adjusted by promoting the interaction between the XLF and Ku80 through protein lactylation. Under the catalysis of GCN5 (lysine acetyltransferase 2A, KAT2A), XLF is lactylated, so that the efficiency of NHEJ is improved, and colorectal cancer (CRC) cells are caused to generate chemotherapy resistance. In addition, inhibition of XLF lactic acidification can increase sensitivity of CRC cells to chemoradiotherapy. A polypeptide is also obtained through design and screening, the polypeptide can specifically inhibit the lactylation of XLF at K288, a synergistic effect is generated on CRC when the polypeptide is combined with 5-FU, and the polypeptide can promote the chemosensitivity of colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to lactic acid modified XLF protein and a preparation and application for inhibiting XLF lactic acidization. Background Art

[0002] DNA double-strand breaks (DSBs) are the most dangerous form of DNA damage and have been shown to cause cell death or trigger neoplastic transformation. In eukaryotic cells, two distinct pathways are primarily responsible for DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). NHEJ is the primary repair mechanism throughout the cell cycle, but because HR requires sister chromatids, it is restricted to S and G2 / M phases. During NHEJ, the Ku70 / 80 heterodimer recognizes and binds to the broken DNA ends. Ku70 / 80 then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the holoenzyme of the DNA-dependent protein kinase (DNA-PK) complex, which functions as a master regulatory kinase and provides a platform for recruiting downstream components of the NHEJ pathway to process DNA ends. During DNA end processing, XRCC4-like factor (XLF) and XRCC4-ligase IV play key roles as scaffolding proteins, bringing two DNA ends into close proximity for DNA end joining. DNA ligase IV interacts with Ku / XRCC4 via one of its BRCT domains. The final step of NHEJ is ligation, mediated by DNA ligase IV. As NHEJ is one of the major DNA repair pathways, altered expression levels of key proteins involved in NHEJ (including Ku70 / 80, DNA-PKcs, Artemis, ligase IV, XRCC4, and XLF) can confer cancer susceptibility. Therefore, directly targeting core components of the DSB repair pathway has become a popular approach to overcome chemotherapy resistance and enhance chemosensitivity in cancer patients.

[0003] Metabolic reprogramming has been identified as one of the ten hallmarks of malignancy. It is characterized by a decrease in mitochondrial oxidative phosphorylation (OXPHOS) and a simultaneous shift to glycolysis for energy compensation, in which pyruvate is used to produce large amounts of lactate catalyzed by lactate dehydrogenase (LDH). Studies have shown that lactate plays an important role in tumorigenesis and chemoresistance, but the detailed mechanisms involved remain unclear. Recent studies have demonstrated that lactate accumulation can dynamically drive lysine lactylation of histone and non-histone proteins, thereby regulating gene expression and protein activity, thereby affecting a variety of physiological and pathological processes, including neuronal development, autophagy, inflammation, cardiovascular disease, and cancer. Recently, studies have reported that lactate-induced lactylation of MRE11 and NBS1 is involved in the overactivation of HR. However, whether protein lactylation regulates other DNA damage repair pathways remains to be determined.

[0004] XRCC4-like factor (XLF, also known as NHEJ1) is located on human chromosome 2q35 and encodes a 299-amino acid protein consisting of an N-terminal globular head domain, a coiled-coil domain that mediates dimerization, and a disordered C-terminal region. Although enzymatically inactive, XLF homodimers serve as essential scaffolding proteins that facilitate interactions between different proteins involved in DNA end synapsis, thereby promoting NHEJ repair. XLF primarily interacts with other proteins through its globular head domain and disordered C-terminal region: the XLF globular head domain interacts with the XRCC4 globular head domain, and the XLF Ku binding motif (X-KBM) in the C-terminal region interacts with Ku70 / 80 via the Ku80 vWA domain. As a core NHEJ factor, abnormal function of XLF is closely associated with tumorigenesis and cancer progression. XLF can promote resistance to oxaliplatin and 5-fluorouracil (5-FU) in colon cancer. Furthermore, oral cancer stem cells with elevated XLF levels are resistant to radiotherapy. However, the underlying mechanism remains to be explored. Summary of the Invention

[0005] The purpose of the present invention is to provide lactated XLF protein and a preparation and application for inhibiting XLF lactation, mainly to solve the technical problem that existing drugs are not effective in treating cancers caused by metabolic abnormalities.

[0006] The technical solutions adopted by the present invention to solve the above technical problems are as follows: The present invention provides the use of lactic acid modified XLF protein as a target in the preparation of a drug for improving tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance, wherein the lactic acid modified site is lysine 288 of the XLF protein.

[0007] As a preferred embodiment, the tumor is colorectal cancer.

[0008] The present invention also provides a preparation combination for improving tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance, wherein the preparation combination includes a preparation for specifically inhibiting the lactic acid modification of XLF protein.

[0009] As a preferred embodiment, the preparation includes small molecule inhibitors, nucleic acids, and polypeptides.

[0010] As a preferred embodiment, the preparation is a polypeptide fused with a cell-penetrating peptide and a sequence shown in SEQ ID NO:4.

[0011] As a preferred embodiment, the sequence of the cell-penetrating peptide is shown in SEQ ID NO: 7. That is, the sequence of the cell-penetrating peptide is HLYVSPWGG; the specific sequence of the polypeptide is: HLYVSPWGGQRPQLSKVKRKKPRG.

[0012] The present invention also provides the use of a reagent for detecting the expression level of lactic acid modification of XLF protein in preparing a kit for predicting the efficacy of tumor chemotherapy, wherein the lactic acid modification site is lysine 288 of the XLF protein.

[0013] As a preferred embodiment, the lactylation modification level of the XLF protein is regulated by lysine acetyltransferase GCN5.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The XLF protein plays a critical role in non-homologous end joining (NHEJ) repair and maintaining genomic stability. This study found that LDHA deficiency inhibits NHEJ. Lactate generated during glycolysis promotes lactylation of XLF at K288 of its Ku binding motif (X-KBM), thereby regulating NHEJ repair efficiency. Mechanistically, DNA damage triggers ATM-mediated phosphorylation of GCN5, which increases GCN5-XLF interaction and XLF lactylation, enhancing XLF-Ku80 binding, XLF recruitment to DSBs, and NHEJ efficiency. Cryo-electron microscopy structural analysis reveals that the X-KBM of lactate (laX-KBM) forms a more extensive interface with Ku70 / 80, inducing conformational changes in the Ku80 vWA domain. Defective XLF lactylation impairs NHEJ and sensitizes cancer cells to chemotherapy. These findings suggest that the GCN5-XLF lactylation axis is a key regulator of NHEJ, and targeting XLF lactylation could improve chemotherapy efficiency.

[0015] This study reports for the first time that protein lactylation regulates the function of XLF in NHEJ by promoting its interaction with Ku80. Lactylation of XLF, catalyzed by GCN5 (lysine acetyltransferase 2A, KAT2A), enhances NHEJ efficiency and confers chemotherapy resistance in colorectal cancer (CRC) cells. Furthermore, inhibition of XLF lactylation increases the sensitivity of CRC cells to chemoradiotherapy.

[0016] The present invention obtains a polypeptide through design and screening, which can specifically inhibit the lactation of XLF at K288 and produce a synergistic effect on CRC when used in combination with 5-FU. The polypeptide can promote the chemotherapy sensitivity of colorectal cancer.

[0017] Our findings reveal a new link between two major cancer hallmarks: metabolic reprogramming and DNA damage response, reveal the important role of protein lactylation in DSB repair, and suggest a new strategy to overcome chemotherapy resistance in CRC patients with high levels of XLF lactylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the EJ5-GFP reporter system of the present invention.

[0019] Figure 2 AK is a data graph showing the lactylation of the NHEJ factor XLF by GCN5 in Example 1 of the present invention.

[0020] Figure 3 AI is a data graph showing the results of Example 2 of the present invention showing that lactylation at K288 of XLF promotes the recruitment of XLF at DSBs.

[0021] Figure 4 AH are the result data diagrams of XLF lactylation regulating NHEJ repair in Example 3 of the present invention.

[0022] Figure 5 AK is a data graph showing the results of Example 4 of the present invention showing that inhibiting the lactylation level of XLF K288 sensitizes colorectal cancer cells to chemotherapy. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described in detail below with reference to the examples. Unless otherwise specified, the reagents and biological materials used below are all commercial products.

[0024] The experimental method process adopted in the present invention is as follows: Co-immunoprecipitation Cell Harvest: Add target cells overexpressing a gene tagged with HA, Flag, or Myc to 4°C pre-chilled PBS and collect them by cell scraping into an EP tube. Cell Lysis: Add 700μl of 1× NETN Cell Lysis Buffer (with protease and phosphatase inhibitors) to each tube of cells (for a 6 cm dish with cells grown to 90% of full growth) and incubate on ice for 30 minutes. Protein Supernatant Collection: Centrifuge the cell lysate at 12,000 rpm for 20 minutes at 4°C to obtain the protein supernatant. Bead Washing: Add 20μl of HA beads to each EP tube, followed by 1ml of 1× NETN Protein Lysis Buffer. Mix thoroughly, centrifuge at 3000 rpm at 4°C for 1 minute, and discard the supernatant. Repeat this step twice. Resuspend and Bind: Remove 80μl of each sample as the input sample. Add the remaining 600μl of protein supernatant to an EP tube containing 20μl of HA beads and incubate on a shaker at 4°C for 2 hours. Wash beads: After incubation, centrifuge at 3000 rpm at 4°C for 1 min, discard the supernatant, then add 1 ml of 1×NETN protein lysis buffer, mix, centrifuge at 3000 rpm at 4°C for 1 min, discard the supernatant, repeat this step 4 times, discard the supernatant, and finally add 50 μl of 1× loading buffer, mix, boil at 100°C for 10 min, centrifuge at 12,000 rpm at 4°C for 3 min, label the samples and store at -20°C.

[0025] Mass spectrometry experiments Sample preparation: prepare 10 175cm 2After 293T cells reached 90% confluence, they were irradiated with 10 Gy and harvested 2 hours after irradiation. Protein extraction: Four volumes of lysis buffer (8 M urea, 3 μM TSA, 50 mM NAM, 1% protease inhibitor) were added and lysed by sonication. Cells were centrifuged at 12,000 g for 10 minutes at 4°C to remove cell debris. The supernatant was transferred to a fresh centrifuge tube and the protein concentration was determined using a BCA assay kit. Trypsin digestion: Equal amounts of protein from each sample were digested, and the volume was adjusted to the same value with lysis buffer. TCA was slowly added to a final concentration of 20%, vortexed to mix, and precipitated at 4°C for 2 hours. Centrifuged at 4,500 g for 5 minutes, the supernatant discarded, and the pellet washed 2-3 times with pre-chilled acetone. After air drying, the pellet was added to a final concentration of 200 mM TEAB and sonicated to disperse the pellet. Trypsin was then added at a 1:50 ratio (protease:protein, m / m) and digested overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mM, and the mixture was reduced at 56°C for 30 min. Iodoacetamide (IAA) was then added to a final concentration of 11 mM, and the mixture was incubated at room temperature in the dark for 15 min. Antibody-based modification enrichment: Peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0). The supernatant was transferred to pre-washed lactate-treated resin (Antibody Resin Product No. PTM-1404, from Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio). The mixture was incubated overnight at 4°C on a rotary shaker with gentle agitation. After incubation, the resin was washed four times with IP buffer and twice with deionized water. Finally, the resin-bound peptides were eluted three times with 0.1% trifluoroacetic acid. The eluate was collected and freeze-dried under vacuum. After drying, desalting was performed according to the instructions of C18 ZipTips, and after vacuum freeze drying, the sample was used for liquid chromatography-mass spectrometry analysis of protein lysine lactylation modification.

[0026] Chromatin fractionation Cells were harvested by scraping, washed once with ice-cold PBS at 4°C, and centrifuged at 1000 rpm for 5 minutes to collect the cell pellet. The pellet was added to extraction buffer 1 (50 mM HEPES pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, protease inhibitors, and phosphatase inhibitors) and lysed on ice for 20 minutes. The pellet was centrifuged at 14,000 g for 3.5 minutes at 4°C to collect the pellet. The pellet was then added to extraction buffer 2 (50 mM HEPES pH 7.5, 1 mM EDTA and 150 mM NaCl, 200 µg / ml RNAse A, protease inhibitors, and phosphatase inhibitors) and incubated with rotation at room temperature for 30 minutes. The pellet was then centrifuged at 14,000 g for 3.5 minutes at 4°C to collect the pellet. 1× SDS protein loading buffer was added, sonicated for 10 seconds, and then boiled at 100°C for 10 minutes. The prepared samples were analyzed for protein levels in chromatin components by western blotting.

[0027] NHEJ repair efficiency assay Cell Seeding: HEK293T cells in logarithmic growth phase were plated in 6-well plates. Plasmid Transfection: When the cells reached approximately 50% confluency, plasmids (m-Cherry: 100ng, ISC: 500ng, NHEJ-GFP: 500ng) were transfected. Six hours later, the culture medium was replaced with normal culture medium. Flow Cytometry: 48 hours after transfection, cells were digested with EDTA-free trypsin and analyzed by flow cytometry. GFP and m-Cherry fluorescence was measured, and NHEJ repair efficiency was calculated ([mCherry + GFP+ / (mCherry+ + mCherry + GFP+)] × 100%).

[0028] Schematic diagram of the EJ5-GFP reporter system Figure 1 As shown: The non-homologous end joining (NHEJ) detection system includes a promoter, puro resistance, and GFP; the promoter is separated from the GFP coding cassette by a puro gene, and the puro gene is flanked by two I-SceI sites with the same orientation. When the I-SceI endonuclease is added, the puro gene is excised, and NHEJ repairs the two I-SceI-induced DSBs. End joining between the I-SceI recognition sites can restore GFP expression.

[0029] CCK8 cell viability assay Cell counting: First, remove the target cells, digest them, and prepare a single-cell suspension. Add 10 μl of the prepared single-cell suspension to a counting chamber, observe under a microscope, and count the cells. Seeding cells: Add the counted cell suspension to a 96-well plate according to the experimental requirements, adding 6,000 cells per well. After the cells have adhered for 24 hours, treat the cells accordingly based on the experimental purpose. Add 20 μl of CCK8 solution to each well and continue incubating in the incubator for 2 hours. Measure the OD450 absorbance value and record it for subsequent analysis.

[0030] Immunofluorescence Seeding cells: First, place coverslips into a 6-well plate and sterilize using UV. Seed the target cells into the same 6-well plate. Allow 24 hours for attachment and then treat the cells according to the experimental purpose. Fixing cells: Aspirate the culture medium, then wash the cells twice with PBS. Fix the cells in the 6-well plate with 4% PFA for 15 minutes at room temperature. Washing cells: Discard the paraformaldehyde and wash the cells three times with PBS for 5 minutes each. Permeabilize the cells with 0.3% Triton X-100 for 5 minutes. Blocking: Block the specimens in the 6-well plate with 1% goat serum for 1 hour. Primary antibody is applied during blocking. Incubation with primary antibody: Aspirate the blocking buffer, add 60 μL of diluted primary antibody, and incubate overnight at 4°C. Incubation with secondary antibody: Wash the cells three times with PBS for 5 minutes each. Apply the immunofluorescence secondary antibody and incubate for 2 hours at room temperature. Mounting: Wash the cells three times with PBS for 5 minutes each. Apply mounting medium containing DAPI and incubate for 2 minutes at room temperature. Finally, wash three times with PBS. Finally, the cells were observed and photographed under a fluorescence microscope for subsequent analysis.

[0031] Laser micro-irradiation-induced DNA damage experiment U2OS cells were cultured overnight in a confocal dish, and then infected with the corresponding virus solution. 10 μM bromodeoxyuridine (BrdU) photosensitizer was added and incubated overnight, followed by laser microirradiation. A laser microirradiation protocol was created: a 365 nm laser (for DNA damage induction) with an intensity of 40% was selected. The laser microirradiation trajectory was selected, and images were captured and saved before DNA damage. After microirradiation, images were captured and saved at the designated time points. All data were analyzed in Image J.

[0032] Comet experiment The prepared cell sample was digested with trypsin and washed twice with PBS, then resuspended in an appropriate amount of PBS to form a single cell suspension. The lysis solution in the kit was pre-cooled at 4°C for at least 20 minutes. The low melting point agarose was then completely dissolved in a 99°C water bath for at least 5 minutes. The hot low melting point agarose was then cooled in a 37°C water bath for at least 20 minutes. The cells were plated at 1×105 Mix the cells / ml with dissolved low-melting-point agarose at a 1:10 volume ratio. Immediately pipette 50µl of the cell mixture onto a slide. Place the slide in a 4°C refrigerator to freeze for 30 minutes, then immerse in lysis solution at 4°C for 40 minutes. Gently add ddH2O to soak the slide twice for 5 minutes each, then add lysis solution and let it sit for 20 minutes. Adjust the level of the electrophoresis buffer to stabilize the power supply at 20V, 200mA, and run the electrophoresis at 4°C for 20 minutes. Gently drain the electrophoresis buffer from the slide, slowly add ddH2O to cover the slide, and let it sit for 3 minutes. Repeat twice, then place it in 70% ethanol and let it sit for 6 minutes. Dry the sample at 37°C for 10 minutes, add one drop of PI solution, cover it, and stain it in the dark for 20 minutes. Observe it under a microscope and photograph it for later analysis.

[0033] Example 1: Core NHEJ factor XLF is lactylated by GCN5 To determine the link between metabolic reprogramming and genomic instability, we first measured the expression of lactate dehydrogenase A (LDHA) and γH2AX in a mouse model of DNA damage repair. We found that Ldha knockout mice developed more γH2AX-positive cells in their lungs and hearts after IR treatment; however, these phenotypes were rescued by treatment with sodium lactate (NALA). Figure 2 A-2C), indicating that the loss of LDHA increases genomic instability. In addition, it was found that inhibition of LDH activity leads to a decrease in cellular NHEJ efficiency (see Figure 2 D). However, sodium lactate treatment directly increases the efficiency of NHEJ (see Figure 2E). These data suggest that lactate can enhance NHEJ repair efficiency. Given that multiple studies have shown that lactate regulates physiological and pathological processes through protein lactylation, this study further explored whether lactate regulates NHEJ repair through protein lactylation. We screened multiple NHEJ proteins and found that XLF exhibited significant lactylation (see Figure 2F). Further investigation revealed that treatment with an LDH inhibitor reduced XLF lactylation, while NALA treatment significantly increased XLF lactylation (see Figures 2G and 2H). We then screened several lactyltransferases (including P300, CBP, KAT5, KAT8, PCAF, and GCN5) for their potential to lactate XLF. Results showed that GCN5 significantly promoted XLF lactylation compared to other lactyltransferases (see Figure 2I). Mass spectrometry analysis revealed that XLF was lactylated at residue K288 (see Figure 2J). To further confirm that the K288 residue of XLF is indeed lactylated upon DNA damage, we generated a K288R mutant. We found that upon DNA damage, lactylation levels increased in XLF-WT, but not in XLF-K288R (see Figure 2K). Taken together, these results indicate that GCN5 is a specific lactyltransferase at the K288 site of XLF.

[0034] Example 2: Lactylation of XLF K288 promotes XLF recruitment at DSBs Given that K288 is located within X-KBM, which is responsible for the interaction between XLF and Ku80 and the recruitment of XLF to DNA damage sites, we found that knocking down GCN5 inhibited the recruitment of XLF to DNA damage sites and its interaction with Ku80 (see Figures 3A-3D). Furthermore, compared with wild-type GFP-XLF, the recruitment rate of the mutant GFP-XLFK288R to DNA damage sites was significantly slower after microirradiation (see Figures 3A-3D). Figure 3 E-3F). Next, the recruitment of XLF to chromatin after DNA damage induction was examined in XLF-WT cells and XLF-K288R mutant cells. The results showed that in wild-type cells, XLF protein levels in the chromatin fraction increased significantly after DNA damage induction; whereas in mutant XLF-K288R cells, XLF protein levels in the chromatin fraction did not change significantly after DNA damage treatment. Furthermore, inhibition of LDH activity after DNA damage resulted in a decrease in the recruitment of XLF WT to chromatin (see Figure 3 G). In addition, treatment with the GCN5 inhibitor MB3 significantly reduced the recruitment of XLF WT to chromatin after DNA damage (see Figure 3H). Meanwhile, sodium lactate (NALA) treatment significantly increased the recruitment of XLF WT to chromatin after DNA damage, but failed to enhance the recruitment of XLF-K288R mutant to chromatin after DNA damage (see Figure 3 G-3H). Next, the interaction between Ku80 and XLF was detected in XLF WT cells and XLF K288R mutant cells. The results showed that the interaction between HA-XLF WT and Ku80 was significantly enhanced under bleomycin and sodium lactate treatment conditions, while the interaction between HA-XLF K288R mutant and Ku80 did not change significantly under bleomycin and sodium lactate treatment conditions ( Figure 3 I). Therefore, the results indicate that GCN5-mediated lactylation at K288 enhances the interaction between XLF and Ku80, thereby promoting the recruitment of XLF to DSBs.

[0035] Example 3: XLF lactylation regulates NHEJ repair Given that XLF recruitment to sites of DNA damage is crucial for the normal progression of NHEJ repair, we next investigated whether XLF lactylation regulates NHEJ repair. We first examined the repair efficiency of HA-XLF wild-type and HA-XLF K288R mutant cells using an NHEJ reporter system. The results showed that compared with XLF WT, the XLF K288R mutant significantly inhibited NHEJ repair. Furthermore, sodium lactate treatment significantly promoted NHEJ repair in cells expressing XLF WT but not the XLF K288R mutant (see ). Figure 4 A). In addition, western blot analysis was performed to examine the levels of γH2AX in wild-type XLF cells and mutant XLF K288R cells at different time points after DNA damage. The results showed that compared with wild-type XLF WT cells, the rate of γH2AX decrease in XLF K288R mutant cells was delayed, indicating that the XLF K288R mutant impedes the DNA damage repair process (see Figure 4 B) In addition, the comet assay was used to further examine the DNA damage and repair in XLF wild-type and XLF K288R mutant cells. The results showed that compared with wild-type XLF cells, XLF K288R mutant cells still had more DNA damage at 8 hours (see Figure 4C and 4D), further demonstrating that lysine lactylation at position 288 of the XLF protein is closely related to NHEJ repair. Next, the chemoradiotherapy sensitivity of XLF WT and XLF K288R mutant colorectal cancer cells was examined. CCK8 assay results showed that HCT116 and SW480 colorectal cancer cells expressing the XLFK288R mutant were more sensitive to 5-FU treatment than HCT116 and SW480 colorectal cancer cells expressing XLF WT (see ). Figure 4 E-4H). Compared with the control group, sodium lactate treatment induced 5-FU resistance in HCT116 and SW480 colorectal cancer cells expressing XLF WT, but not in HCT116 and SW480 colorectal cancer cells expressing the XLF K288R mutant (see Figure 4 E and 4F). In contrast, treatment with the GCN5 inhibitor (MB-3) sensitized HCT116 and SW480 colorectal cancer cells expressing XLF WT but not the XLFK288R mutant to 5-FU (see Figure 4 G and 4H). Taken together, these results indicate that GCN5-mediated lactylation of XLF at K288 is critical for NHEJ repair and colorectal cancer cell survival after chemotherapy-induced DNA damage.

[0036] Example 4: Inhibition of XLF K288 lactylation sensitizes colorectal cancer cells to chemotherapy Since targeting GCN5 or LDH affects multiple signaling pathways, it may have significant side effects. The strategy of generating peptides based on the amino acid sequences involved in protein post-translational modifications has been proven to be an effective research method for inhibiting protein post-translational modifications. In order to more specifically target XLF K288 lactylation, we generated six peptides fused with cell-penetrating peptides (CPPs) based on the sequence of XLFK288 lactylation (see Figure 5 A). See Table 1 for sequence information. The sequence of cell-penetrating peptide CPPs is shown in SEQ ID NO:7. SEQ ID NO:1 to SEQ ID NO:6 are short peptides at the K288 site of XLF. However, the six designed short peptides are not lactylated at the corresponding K288 sites. Cell-penetrating peptide CPPs are fused with the six short peptides to obtain Figure 5 Peptide sequences of Pep1-Pep6 in A.

[0037]

[0038] Next, HEK293T cells transfected with HA-XLF were treated with the control peptide XLF K288R (Pep1) and XLF K288 peptides (Pep2 to Pep6). After screening, it was found that only Pep4 (XLF K288) (hereinafter referred to as Pep4) had a significant effect on inhibiting XLF K288 lactylation (see Figure 5 Furthermore, Pep4 significantly reduced the lactation level of XLFWT compared to the control, but had no significant effect on the lactation level of the XLF K288R mutant (see Figure 5 D). Next, we examined the effect of XLF K288 peptide on DNA damage-induced XLF recruitment to DNA damage sites and its interaction with Ku80. We found that after DNA damage, Pep4 reduced the binding of Ku80 to XLF (see Figure 5 E). Compared with the control group, Pep4 inhibited the binding of XLF WT to chromatin after DNA damage; compared with the control group, Pep4 had no significant effect on the chromatin recruitment of XLF K288R mutant after DNA damage (see Figure 5 F). The effect of Pep4 on cellular NHEJ repair was then examined. The results showed that compared with the control group, Pep4-treated cells significantly inhibited NHEJ, and sodium lactate supplementation did not rescue the decrease in NHEJ caused by Pep4, while the control peptide Pep1 did not have this phenomenon (see Figure 5 G). These results suggest that Pep4 (XLF K288) can inhibit XLF recruitment to DNA damage sites and its interaction with Ku80 by inhibiting XLF lactylation, thereby hindering NHEJ repair. Next, the role of Pep4 in the response to chemoradiotherapy in colorectal cancer cells was further explored. CCK8 assay results showed that Pep4 treatment, but not Pep1, sensitized colorectal cancer cell lines HCT116 to 5-FU treatment compared to the control group (see Figure 5 H). In addition, Pep4 increased the chemosensitivity of colorectal cancer cells to 5-FU compared with the control group in HCT116 cells expressing XLF WT but not XLF K288R (see Figure 5 I). To further confirm the effect of Pep4 on the response to chemotherapy in colorectal cancer, an in vivo tumor killing assay was performed using a colorectal cancer patient-derived xenograft (PDX) model.

[0039] The human colorectal cancer xenograft (PDX) model method is as follows: Tumor tissue (derived from colorectal cancer patients) stored in liquid nitrogen was revived and then placed on ice and brought to the animal room for the following procedures. The resuscitation steps were the same as those for cell resuscitation. First, athymic nude mice were anesthetized with isoflurane, and then small pieces of tumor measuring 1*1*1mm were inoculated on both sides of the nude mice's abdomen and back. When the tumor tissue grew to 600mm, 3 At about 30 seconds, the mice were killed by dislocating the neck, and the tumor tissue was removed and placed on ice. It was cut into small tumor pieces of 1*1*1mm and washed three times with PBS. It was divided into two parts, one part was added with freezing solution and frozen in liquid nitrogen. The other part was used to construct the next generation PDX model. The tumor pieces were inoculated on both sides of the abdomen and back of the nude mice. One week after inoculation, the growth of the tumor could be obviously observed. The nude mice were randomly divided into 6 groups: experimental control group, Pep1 treatment group, Pep4 treatment group, 5-FU treatment group, 5-FU and Pep1 combined treatment group, 5-FU and Pep4 combined treatment group. 5-FU was administered by intraperitoneal injection every other day at a dose of 10 mg / kg. Pep4 or Pep1 was administered by intraperitoneal injection every day at a dose of 10 mg / kg. During this period, the status of the mice was monitored every day, and the tumor size was measured once every two days with a vernier caliper, and the long and short axis data of the tumor were recorded. When the tumor tissue grows to 600mm 3 At about 4 h, the mice were killed by cervical dislocation, and the tumor tissues were removed, photographed, and weighed. At the same time, the tumor tissues were divided into two parts, one was stored in liquid nitrogen, and the other was fixed with 4% paraformaldehyde and stored at 4 degrees, and then embedded, sliced, and stained.

[0040] First, we screened the lactylation modification levels of XLF in three colorectal cancer PDX samples. The results showed that the lactylation modification level of XLF in PDX sample No. 1 was the highest (see Figure 5 J). Next, PDX sample No. 1 was used to construct a PDX model in nude mice for in vivo tumor killing experiments. The in vivo results showed that Pep1 and Pep4 had no significant effect on the tumor size and weight of colorectal cancer PDXs, indicating that Pep1 and Pep4 do not affect the proliferation of colorectal cancer PDXs. However, Pep4, but not Pep1, significantly increased the sensitivity of colorectal cancer PDXs to 5-FU treatment (see Figure 5 These findings suggest that Pep4-targeted lactylation of XLF K288 impairs XLF chromatin recruitment and interaction with Ku80, thereby promoting chemosensitivity in colorectal cancer.

[0041] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the scope of protection of the present invention.

Claims

1. Use of lactic acid-modified XLF protein as a target in the preparation of drugs for enhancing tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance, characterized in that: The lactylation modification site is lysine 288 of the XLF protein.

2. The use according to claim 1, characterized in that: The tumor is colorectal cancer.

3. A preparation combination for enhancing tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance, characterized in that: The invention comprises a preparation that specifically inhibits the lactylation modification of XLF protein.

4. The preparation combination for improving tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance according to claim 3, characterized in that: The preparations include small molecule inhibitors, nucleic acids, and polypeptides.

5. The preparation combination for improving tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance according to claim 3, characterized in that: The preparation is a polypeptide fused with a cell-penetrating peptide and a sequence shown in SEQ ID NO:

4.

6. The preparation combination for enhancing tumor chemotherapy sensitivity or reversing tumor chemotherapy resistance according to claim 5, characterized in that: The sequence of the cell-penetrating peptide is shown in SEQ ID NO:

7.

7. Use of a reagent for detecting the expression level of lactic acid modification of XLF protein in the preparation of a kit for predicting the efficacy of tumor chemotherapy, characterized in that: The lactylation modification site is lysine 288 of the XLF protein.

8. The use according to claim 7, characterized in that: The lactylation modification level of the XLF protein is regulated by lysine acetyltransferase GCN5.

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