Lactylation-modified xlf protein and preparation and application for inhibiting xlf lactylation

Abstract

The present application relates to a preparation and application of a lactylation modified XLF protein and inhibition of XLF lactylation. The present application researches and finds that protein lactylation regulates the function of XLF in the NHEJ process by promoting the interaction of XLF and Ku80. Under the catalysis of GCN5 (lysine acetyltransferase 2A, KAT2A), XLF is lactylated to improve the efficiency of NHEJ and cause the chemotherapeutic resistance of colorectal cancer (CRC) cells. In addition, the inhibition of XLF lactylation can increase the sensitivity of CRC cells to chemotherapy and radiotherapy. The present application also obtains a polypeptide by design and screening, which can specifically inhibit the lactylation of XLF at K288, and has a synergistic effect on CRC when combined with 5-FU, and the polypeptide can promote the chemotherapeutic sensitivity of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to lactation-modified XLF protein and preparations for inhibiting XLF lactation and their applications. Background Technology

[0002] DNA double-strand breaks (DSBs) are the most dangerous form of DNA damage, proven to lead to cell death or trigger tumor transformation. In eukaryotic cells, two distinct pathways are primarily responsible for DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways. NHEJ is the dominant repair mechanism throughout the cell cycle, while HR, requiring sister chromatids, is limited to the S and G2 / M phases. During NHEJ, the Ku70 / 80 heterodimer recognizes and binds to the broken DNA ends. Ku70 / 80 then recruits DNA-dependent protein kinase catalytic subunits (DNA-PKcs) to form the holoenzyme of the DNA-dependent protein kinase (DNA-PK) complex, which plays a major regulatory role in the kinase and provides a platform for recruiting downstream components of the NHEJ pathway for DNA end treatment. In DNA end processing, XRCC4-like factor (XLF) and XRCC4-ligase IV play crucial roles as scaffold proteins, bringing two DNA ends closer together for DNA end ligation. DNA ligase IV interacts with Ku / XRCC4 through one of its BRCT domains. The final step in NHEJ is ligation mediated by DNA ligase IV. Since 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 lead to cancer susceptibility. Therefore, directly targeting the core components of the DSB repair pathway has become a common approach to overcome chemotherapy resistance and improve chemosensitivity in cancer patients.

[0003] Metabolic reprogramming has been identified as one of the top ten hallmarks of malignant tumors. It is characterized by reduced mitochondrial oxidative phosphorylation (OXPHOS) and energy compensation through glycolysis, in which pyruvate is used to produce large amounts of lactate via lactate dehydrogenase (LDH). Studies have shown that lactate plays a crucial role in tumorigenesis and chemotherapeutic resistance, but the detailed mechanisms involved remain unclear. Recent research indicates that lactate accumulation can dynamically drive histone and non-histone lysine lactation, thereby regulating gene expression and protein activity, and consequently affecting a variety of physiological and pathological processes, including neuronal development, autophagy, inflammation, cardiovascular disease, and cancer. Recently, studies have reported that lactate-induced MRE11 and NBS1 lactation is involved in the overactivation of heart rate (HR). However, whether protein lactation 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 containing an N-terminal globular head domain, a coiled domain that mediates dimerization, and a disordered C-terminal region. Although lacking enzymatic activity, the XLF homodimer is an essential scaffold protein that promotes interactions between different proteins involved in DNA terminal synapses, thereby facilitating 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 XLF function is closely associated with tumorigenesis and cancer progression. XLF can promote resistance to oxaliplatin and 5-fluorouracil (5-FU) in colorectal cancer. Furthermore, oral cancer stem cells with elevated XLF levels are resistant to radiotherapy. However, the underlying mechanisms remain to be explored. Summary of the Invention

[0005] The purpose of this invention is to provide lactation-modified XLF protein and formulations for inhibiting XLF lactation, as well as their applications. It primarily addresses the technical problem of poor therapeutic efficacy of existing drugs for cancers caused by metabolic abnormalities.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides the application of lactolyzed XLF protein as a target in the preparation of drugs that improve the sensitivity of tumors to chemotherapy or reverse tumor resistance to chemotherapy, wherein the lactolyzed site is lysine 288 of the XLF protein.

[0008] As a preferred embodiment, the tumor is colorectal cancer.

[0009] The present invention also provides a formulation combination for improving tumor chemosensitivity or reversing tumor chemosensitivity, the formulation combination comprising a formulation that specifically inhibits the lactation modification of XLF protein.

[0010] As a preferred embodiment, the formulation includes small molecule inhibitors, nucleic acids, and peptides.

[0011] As a preferred embodiment, the formulation is a polypeptide fused with a cell-penetrating peptide and the sequence shown in SEQ ID NO:4.

[0012] 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 sequence of the polypeptide is specifically: HLYVSPWGGQRPQLSKVKRKKPRG.

[0013] The present invention also provides the application of a reagent for detecting the expression level of lactation modification of XLF protein in the preparation of a kit for predicting the efficacy of tumor chemotherapy, wherein the lactation modification site is lysine 288 of XLF protein.

[0014] In a preferred embodiment, the lactation modification level of the XLF protein is regulated by lysine acetyltransferase GCN5.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] XLF proteins play a crucial role in non-homologous end join (NHEJ) repair and maintaining genome stability. This study found that LDHA deficiency inhibits NHEJ. Lactic acid produced during glycolysis promotes the lactation of XLF at the K288 site of its Ku-binding motif (X-KBM), thereby regulating NHEJ repair efficiency. Mechanistically, DNA damage triggers ATM-mediated GCN5 phosphorylation, increasing GCN5-XLF interaction and XLF lactation, enhancing XLF-Ku80 binding, XLF recruitment to the DSB, and NHEJ efficiency. Cryo-electron microscopy structural analysis revealed that the lactate X-KBM (laX-KBM) forms a broader interface with Ku70 / 80, inducing conformational changes in the Ku80 vWA domain. Defective XLF lactation impairs NHEJ and sensitizes cancer cells to chemotherapy. These findings suggest that the GCN5-XLF lactation axis is a key regulator of NHEJ, and targeting XLF lactation can improve chemotherapy efficacy.

[0017] This invention reports for the first time that protein lactation regulates the function of XLF in the NHEJ process by promoting the interaction between XLF and Ku80. XLF is lactated under the catalysis of GCN5 (lysine acetyltransferase 2A, KAT2A), thereby increasing the efficiency of NHEJ and leading to chemoresistance in colorectal cancer (CRC) cells. Furthermore, inhibiting XLF lactation increases the sensitivity of CRC cells to chemoradiotherapy.

[0018] This invention designs and screens a polypeptide that specifically inhibits the lactation of XLF at K288 and has a synergistic effect on CRC when used in combination with 5-FU. This polypeptide can promote the chemosensitivity of colorectal cancer.

[0019] The findings of this invention reveal a new link between two major cancer features (metabolic reprogramming and DNA damage response), reveal the important role of protein lactation in DSB repair, and propose a new strategy to overcome chemotherapy resistance in CRC patients with high levels of XLF lactation. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the EJ5-GFP reporting system in this invention.

[0021] Figure 2 AK is a graph showing the results of lactation of NHEJ factor XLF by GCN5 in Example 1 of this invention.

[0022] Figure 3 AI is a graph showing the results of lactation at K288 of XLF promoting recruitment of XLF in DSB in Example 2 of this invention.

[0023] Figure 4 AH is a graph showing the results of XLF lactation regulating NHEJ repair in Example 3 of this invention.

[0024] Figure 5 AK is a graph showing the results of inhibiting the lactation level of XLF K288 in Example 4 of this invention, which made colorectal cancer cells sensitive to chemotherapy. Detailed Implementation

[0025] The technical solution of the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, all reagents and biological materials used below are commercial products.

[0026] The experimental method used in this invention is as follows:

[0027] Immunoprecipitation

[0028] Cell Collection: Overexpressing target cells tagged with HA, Flag, or Myc were added to pre-chilled PBS at 4°C and collected into EP tubes using a cell scraper. Cell Lysis: 700 μl of 1×NETN cell lysis buffer (containing protease and phosphatase inhibitors) was added to each tube of cells (cells in a 6 cm dish reaching 90% confluence), and the cells were incubated on ice for 30 minutes for lysis. Protein Supernatant Collection: The cell lysis buffer was centrifuged at 12,000 rpm for 20 minutes at 4°C to obtain the protein supernatant. Bead Washing: 20 μl of HA beads were added to each EP tube, followed by 1 ml of 1×NETN protein lysis buffer. After mixing, the tubes were centrifuged at 3000 rpm for 1 minute at 4°C, and the supernatant was discarded. This step was repeated twice. Resuspension: 80 μl of each sample was reserved as the input sample. The remaining 600 μl of protein supernatant was added to an EP tube containing 20 μl of HA beads, and the tubes were incubated on a shaker at 4°C for 2 hours. Washing beads: After incubation, centrifuge at 3000 rpm for 1 min at 4°C, discard the supernatant, then add 1 ml of 1×NETN protein lysis buffer, mix well, centrifuge at 3000 rpm for 1 min at 4°C, discard the supernatant, repeat this step 4 times, then discard the supernatant, finally add 50 μl of 1×loading buffer, mix well, boil at 100°C for 10 min, centrifuge at 4°C for 3 min at 12,000 rpm, label the sample and store at -20°C.

[0029] Mass spectrometry experiment

[0030] Sample preparation: Prepare 10 samples of 175cm in height. 2293T cells that had reached 90% confluence were irradiated with 10 Gy, and collected 2 hours later. Protein extraction: Lysis was performed by sonication after adding 4 times the volume of lysis buffer (8 M urea, 3 μM TSA, 50 mM NAM, 1% protease inhibitor). The cells were centrifuged at 12000 g for 10 min at 4°C to remove cell debris. The supernatant was transferred to a new centrifuge tube, and protein concentration was determined using a BCA assay kit. Trypsin digestion: Equal volumes of protein from each sample were digested, and the volumes were adjusted to be uniform with lysis buffer. A final concentration of 20% TCA was slowly added, vortexed, and incubated at 4°C for 2 h. The cells were centrifuged at 4500 g for 5 min, the supernatant was discarded, and the precipitate was washed 2-3 times with pre-cooled acetone. After drying, 200 mM TEAB was added, the precipitate was sonicated, and trypsin was added at a 1:50 ratio (protease: protein, m / m), and digestion was performed overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mM, and reduction was performed at 56 °C for 30 min. Iodoacetamide (IAA) was then added to a final concentration of 11 mM, and incubation was carried out at room temperature in the dark for 15 min. Antibody-based enrichment: Peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0), and the supernatant was transferred to pre-washed lactated resin (antibody resin catalog number PTM-1404, sourced from Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio). The resin was placed on a rotary shaker at 4 °C, gently shaken, and incubated overnight. After incubation, the resin was washed four times with IP buffer and twice with deionized water. Finally, the resin-bound peptides were eluted using 0.1% trifluoroacetic acid elution buffer, for a total of three elutions. The eluent was collected and freeze-dried under vacuum. After drying, the sample was desalted according to the C18 ZipTips instruction manual, and then vacuum freeze-dried for analysis of protein lysine lactation modification by liquid chromatography-mass spectrometry.

[0031] Chromatin component separation

[0032] Cells were collected using a cell scraper, washed once with pre-cooled PBS at 4°C, and centrifuged at 1000 rpm for 5 minutes to collect the cell pellet. The collected cell 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 inhibitor, phosphatase inhibitor) and lysed on ice for 20 minutes. The pellet was then centrifuged at 14,000 g at 4°C for 3.5 minutes, collected, and added to Extraction Buffer 2 (50 mM HEPES pH 7.5, 1 mM EDTA and 150 mM NaCl, 200 µg / ml RNase A, protease inhibitor, phosphatase inhibitor) and incubated at room temperature for 30 minutes. The pellet was then centrifuged at 14,000 g at 4°C for 3.5 minutes, collected, and added to 1×SDS protein loading buffer. The pellet was sonicated for 10 seconds and then boiled at 100°C for 10 minutes. The prepared samples were analyzed using Western blotting to detect the protein levels of chromatin components.

[0033] NHEJ Repair Efficiency Test

[0034] Cell seeding: HEK293T cells in logarithmic growth phase were seeded in 6-well plates. Transfection with plasmids: When cell confluence reached approximately 50%, plasmids (m-Cherry: 100 ng, ISC: 500 ng, NHEJ-GFP: 500 ng) were transfected. After 6 hours, the medium was replaced with normal medium. Flow cytometry: 48 hours post-transfection, cells were digested with EDTA-free trypsin and analyzed by flow cytometry. GFP and m-Cherry fluorescence were detected, and the NHEJ repair efficiency was calculated as ([mCherry + GFP+ / (mCherry+ + mCherry + GFP+)] × 100%).

[0035] A schematic diagram of the EJ5-GFP reporter system is shown below. Figure 1 As shown: The non-homologous end joining (NHEJ) detection system contains a promoter, purine resistance, and GFP. The promoter is separated from the GFP coding cassette by a puro gene. The puro gene is flanked by two I-SceI sites with the same orientation. When I-SceI endonuclease is added, the puro gene is excised, and NHEJ repairs the two I-SceI-induced DSBs. The end joining between the I-SceI recognition sites can restore GFP expression.

[0036] CCK8 cell viability assay

[0037] Cell counting: First, target cells were collected, digested, and a single-cell suspension was prepared. 10 μl of the prepared single-cell suspension was added to a counting chamber, and the cells were observed and counted under a microscope. Cell seeding: The counted cell suspension was added to 96-well plates according to experimental requirements, with 6000 cells per well. After 24 hours of cell attachment, the cells were treated accordingly based on the experimental objectives. 20 μl of CCK8 solution was added to each well, and the cells were incubated for another 2 hours. The OD450 absorbance was measured and recorded for subsequent analysis.

[0038] Immunofluorescence

[0039] Cell Seeding: First, place coverslips into 6-well plates and sterilize them using a UV sterilizer. Seed the target cells into the 6-well plates and allow them to adhere for 24 hours before processing according to the experimental objectives. Cell Fixation: Aspirate the culture medium, then wash the cells with PBS, repeating twice. Fix the cells in the 6-well plates with 4% PFA at room temperature for 15 minutes. Cell Washing: Discard the paraformaldehyde, then wash the cells three times with PBS, 5 minutes each time. Cell Penetration: Penetrate the cells with 0.3% Triton X-100 for 5 minutes. Blocking: Add 1% sheep serum to the 6-well plates to block the specimen for 1 hour. Prepare the primary antibody for blocking. Primary Antibody Incubation: Aspirate the blocking buffer, add 60 μl of diluted primary antibody, and incubate overnight at 4°C. Secondary Antibody Incubation: Wash the cells three times with PBS, 5 minutes each time, add immunofluorescence secondary antibody, and incubate at room temperature for 2 hours. Mounting: Wash the cells three times with PBS, 5 minutes each time, add mounting medium containing DAPI, incubate at room temperature for 2 minutes, and finally wash three times with PBS. Finally, the images were observed and photographed under a fluorescence microscope for subsequent analysis.

[0040] Laser micro-radiation induced DNA damage experiment

[0041] U2OS cells were seeded and cultured overnight in confocal dishes, then infected with the appropriate viral solution. After incubation overnight with 10 μM bromideoxyuridine (BrdU) photosensitizer, laser micro-irradiation experiments were performed. A laser micro-irradiation workflow was created: a 365nm laser with an intensity of 40% was selected for DNA damage induction; the laser micro-irradiation trajectory was chosen; and images before DNA damage were acquired and saved. After micro-irradiation, images were acquired and saved at specified time points. All data were analyzed in ImageJ.

[0042] Comet Experiment

[0043] Digest the prepared cell samples with trypsin, wash twice with PBS, and then resuspend in an appropriate amount of PBS to form a single-cell suspension. Pre-cool the lysis buffer from the kit at 4°C for at least 20 minutes. Then completely dissolve the low-melting-point agarose in a 99°C water bath for at least 5 minutes. Next, cool the heated low-melting-point agarose in a 37°C water bath for at least 20 minutes. Spread the cells at a concentration of 1×10⁻⁶ cells / mL. 5 Mix the cell mixture with dissolved low-melting-point agarose at a ratio of 1:10 (v / v) until homogeneous, and immediately pipette 50 µl of the cell mixture onto a glass slide. Freeze the slide at 4°C for 30 minutes, then immerse it in lysis solution at 4°C for 40 minutes. Gently add ddH₂O twice, soaking the slide for 5 minutes each time, then add unwinding solution and let it stand for 20 minutes. Adjust the electrophoresis buffer level to stabilize the power supply parameters at 20V, 200mA, and 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 stand for 3 minutes. Repeat twice. After the electrophoresis, let it stand in 70% ethanol for 6 minutes. Dry the sample at 37°C for 10 minutes, add 1 drop of PI solution, cover with a coverslip, stain in the dark for 20 minutes, then observe and photograph the image under a microscope for subsequent analysis.

[0044] Example 1: Core NHEJ factor XLF is lactated by GCN5

[0045] To determine the link between metabolic reprogramming and genomic instability, the expression of lactate dehydrogenase A (LDHA) and γH2AX in a DNA damage repair mouse model was first measured. The results showed that LdHA knockout mice exhibited more γH2AX-positive cells in their lung and heart tissues after IR treatment; however, treatment with sodium lactate (NALA) rescued these phenotypes (see [link to study]). Figure 2 (A-2C), indicating that the absence of LDHA increases genomic instability. Furthermore, it was found that inhibiting LDH activity leads to a decrease in cellular NHEJ efficiency (see A-2C). Figure 2 D). However, sodium lactate treatment directly improved the efficiency of NHEJ (see D). Figure 2E). These data indicate that lactate can improve the efficiency of NHEJ repair. Given that multiple studies have shown that lactate regulates physiological and pathological processes through protein lactation, this invention further explores whether lactate regulates NHEJ repair through protein lactation. We screened several NHEJ proteins and found that XLF has a significant lactation effect (see Figure 2F). Further studies revealed that LDH inhibitor treatment led to a decrease in XLF lactation, while NALA treatment significantly increased XLF lactation (see Figures 2G and 2H). Next, we screened the potential of several lactate transferases (including P300, CBP, KAT5, KAT8, PCAF, and GCN5) to lactate XLF. The results showed that GCN5 promoted XLF lactation more strongly than other lactate transferases (see Figure 2I). Mass spectrometry analysis showed that XLF underwent lactation at residue K288 (see Figure 2J). To further confirm that the K288 residue of XLF is indeed lactated after DNA damage, we performed a mutation, generating the K288R mutant. The study found that the lactation level of XLF-WT increased after DNA damage, while that of XLF-K288R did not (see Figure 2K). In summary, these results indicate that GCN5 is a specific lactate transferase at the XLF K288 site.

[0046] Example 2: Lactic acidification at K288 of XLF promotes the recruitment of XLF to the DSB.

[0047] Given that K288 is located within the 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 inhibits the recruitment of XLF at DNA damage sites and its interaction with Ku80 (see Figures 3A-3D). Furthermore, compared to wild-type GFP-XLF, the microradiation-treated mutant GFP-XLFK288R showed a significantly slower recruitment rate at DNA damage sites (see Figure 3A-3D). Figure 3 E-3F). Next, the recruitment of XLF in chromatin after DNA damage induction was examined in XLF-WT cells and XLF-K288R mutant cells. The results showed that in wild-type cells, the level of XLF protein in the chromatin fraction significantly increased after DNA damage induction; while in mutant XLF-K288R cells, the level of XLF protein in the chromatin fraction did not change significantly after DNA damage treatment, and inhibition of LDH activity after DNA damage led to a decrease in XLF WT recruitment in chromatin (see E-3F). Figure 3 G). Furthermore, treatment with the GCN5 inhibitor MB3 significantly reduced the recruitment of XLF WT into chromatin following DNA damage (see [link]). Figure 3 H). Meanwhile, sodium lactate (NALA) treatment significantly increased chromatin recruitment of XLF WT after DNA damage, but failed to enhance chromatin recruitment of the XLF-K288R mutant after DNA damage (see [link to relevant documentation]). Figure 3 G-3H). Next, the interaction between Ku80 and XLF was examined 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, while the interaction between HA-XLF K288R mutant and Ku80 did not change significantly under bleomycin and sodium lactate treatment. Figure 3 I). Therefore, the results indicate that GCN5-mediated lactation at K288 enhances the interaction between XLF and Ku80, thereby promoting XLF recruitment to DSB sites.

[0048] Example 3: XLF lactation regulates NHEJ repair

[0049] Given that the recruitment of XLF at DNA damage sites plays a crucial role in the normal progression of NHEJ repair, we next investigated whether XLF lactation regulates NHEJ repair. First, the repair efficiency of HA-XLF wild-type and HA-XLF K288R mutant cells was detected using the NHEJ reporter system. The results showed that, compared with XLF WT, the XLF K288R mutant significantly inhibited NHEJ repair; simultaneously, in cells expressing XLF WT but not the XLF K288R mutant, sodium lactate treatment significantly promoted NHEJ repair (see [link to article]). Figure 4 A). Furthermore, the levels of γH2AX in wild-type XLF cells and mutant XLF K288R cells at different time points after DNA damage were detected by Western blot. The results showed that compared with wild-type XLF WT cells, XLF K288R mutant cells delayed the rate of γH2AX decrease, indicating that the XLF K288R mutant inhibited the DNA damage repair process (see [link]). Figure 4 (B) Furthermore, the DNA damage repair in XLF wild-type and XLF K288R mutant cells was further examined using a comet assay. The results showed that compared with wild-type XLF cells, XLF K288R mutant cells still had more DNA damage after 8 hours (see B). Figure 4(C and 4D) further demonstrate that lysine lactation at position 288 of the XLF protein is closely related to NHEJ repair. Next, the chemoradiosensitivity of XLF WT and XLF K288R mutant colorectal cancer cells was examined. CCK8 assay results showed that, compared with HCT116 and SW480 colorectal cancer cells expressing XLF WT, HCT116 and SW480 colorectal cancer cells expressing the XLF K288R mutant were more sensitive to 5-FU treatment (see [link to C and 4D]). Figure 4 E-4H). Furthermore, compared to the control group, sodium lactate treatment induced resistance to 5-FU in HCT116 and SW480 colorectal cancer cells expressing XLF WT, but did not induce resistance to 5-FU in XLF K288R mutant HCT116 and SW480 colorectal cancer cells (see [link to original text]). Figure 4 E and 4F). Conversely, treatment with the GCN5 inhibitor (MB-3) made HCT116 and SW480 colorectal cancer cells expressing the XLF WT mutant, rather than the XLFK288R mutant, more sensitive to 5-FU (see E and 4F). Figure 4 (G and 4H). In summary, these results indicate that GCN5-mediated lactation of XLF at K288 is crucial for NHEJ repair and colorectal cancer cell survival following chemotherapy-induced DNA damage.

[0050] Example 4: Inhibiting XLF K288 lactation levels to sensitize colorectal cancer cells to chemotherapy

[0051] Targeting GCN5 or LDH can affect multiple signaling pathways and may produce significant side effects. Strategies for specifically generating peptides based on the amino acid sequences involved in protein post-translational modifications have proven to be an effective research method for inhibiting protein post-translational modifications. To more specifically target XLF K288 lactation, we generated six peptides fused with cell-penetrating peptides (CPPs) based on the XLFK288 lactation sequence (see [link to article]). Figure 5 A). Sequence information is shown in Table 1. The sequence of the 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 were not lactated at the corresponding K288 sites. The cell-penetrating peptide CPPs were fused with the six short peptides to obtain the desired result. Figure 5 The polypeptide sequences of Pep1-Pep6 in A.

[0052]

[0053] Next, HEK293T cells transfected with HA-XLF were treated with the control peptide XLF K288R (Pep1) and XLF K288 polypeptides (Pep2-Pep6). After screening, only Pep4 (XLF K288) (hereinafter referred to as Pep4) was found to have a significant effect on inhibiting XLF K288 lactation (see [link to Pep4]). Figure 5 B and 5C). Furthermore, compared to the control group, Pep4 significantly reduced lactation levels in XLFWT but had no significant effect on lactation levels in the XLF K288R mutant (see [link to relevant documentation]). Figure 5 D). Next, we examined the effect of the XLF K288 peptide on DNA damage-induced recruitment of XLF at DNA damage sites and its interaction with Ku80. We found that after DNA damage, Pep4 reduced the binding of Ku80 to XLF (see [link to relevant documentation]). Figure 5 E). Simultaneously, compared to the control group, Pep4 inhibited the binding of XLF WT to chromatin after DNA damage; compared to the control group, Pep4 had no significant effect on chromatin recruitment in the XLF K288R mutant after DNA damage (see [link to relevant documentation]). Figure 5 F). Next, the effect of Pep4 on cellular NHEJ repair was examined. The results showed that, compared with the control group, Pep4 treatment significantly inhibited NHEJ, and sodium lactate supplementation did not salvage the NHEJ decrease induced by Pep4, while the control peptide Pep1 did not exhibit this phenomenon (see [link to relevant documentation]). Figure 5 These results indicate that Pep4 (XLF K288) can inhibit XLF recruitment at DNA damage sites and its interaction with Ku80 by suppressing XLF lactation, thereby hindering NHEJ repair. Next, the effect of Pep4 on the chemoradiotherapy response of colorectal cancer cells was further investigated. CCK8 assay results showed that, compared with the control group, Pep4 treatment instead of Pep1 made HCT116 colorectal cancer cells sensitive to 5-FU treatment (see [link to CCK8 assay]). Figure 5 Furthermore, in HCT116 cells expressing XLF WT but not XLF K288R, Pep4 increased the chemosensitivity of colorectal cancer cells to 5-FU compared to the control group (see H). Figure 5 I). To further confirm the effect of Pep4 on chemotherapy response in colorectal cancer, in vivo tumor killing experiments were conducted using a patient-derived xenograft (PDX) model of colorectal cancer.

[0054] The method for human colorectal cancer xenograft (PDX) model is as follows:

[0055] Tumor tissue (derived from colorectal cancer patients) preserved in liquid nitrogen was resuscitated and then transported to the animal facility on ice for the following procedures. The resuscitation steps were the same as for cell resuscitation. Athymic nude mice were first anesthetized with isoflurane, and then 1*1*1 mm tumor fragments were inoculated onto both sides of the abdomen and back of the mice. The tumor tissue was allowed to grow to 600 mm. 3 At approximately 10:00 AM, mice were euthanized by cervical dislocation. Tumor tissue was removed and placed on ice, then cut into 1*1*1 mm pieces and washed three times with PBS. The pieces were then divided into two portions; one portion was cryopreserved in liquid nitrogen. The other portion was used to construct a next-generation PDX model. The tumor pieces were inoculated into the abdomen and back of nude mice, and tumor growth was clearly observed one week after inoculation. Nude mice were randomly divided into six groups: experimental control group, Pep1 treatment group, Pep4 treatment group, 5-FU treatment group, 5-FU and Pep1 combined treatment group, and 5-FU and Pep4 combined treatment group. 5-FU was administered intraperitoneally at a dose of 10 mg / kg every other day. Pep4 or Pep1 was administered intraperitoneally daily at a dose of 10 mg / kg. During this period, the mice's condition was monitored daily, and tumor size was measured every two days using calipers, recording the long and short axes of the tumor. Tumors were allowed to grow to 600 mm in length. 3 At approximately 10:00 AM, the mice were euthanized by cervical dislocation, and the tumor tissue was removed, photographed, recorded, and weighed. The tumor tissue was divided into two parts: one part was stored in liquid nitrogen, and the other part was fixed with 4% paraformaldehyde and stored at 4 degrees Celsius. Later, it was embedded, sectioned, and stained.

[0056] First, the lactation modification level of XLF in three colorectal cancer PDX samples was screened. The results showed that the highest lactation modification level of XLF was found in PDX sample No. 1 (see...). Figure 5 J). Next, a PDX model was constructed in nude mice using PDX sample 1 for in vivo tumor killing experiments. In vivo results showed that Pep1 and Pep4 had no significant effect on the tumor size and weight of colorectal cancer PDX, indicating that Pep1 and Pep4 do not affect the proliferation of colorectal cancer PDX. However, Pep4, but not Pep1, significantly improved the sensitivity of colorectal cancer PDX to 5-FU treatment (see [link to relevant documentation]). Figure 5 These findings suggest that Pep4-targeted lactation of XLF K288 weakens the recruitment of XLF chromatin and its interaction with Ku80, thereby promoting chemosensitivity in colorectal cancer.

[0057] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of these embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the present invention's technical solutions, and any such changes and modifications are within the protection scope of the present invention.

Claims

1. The application of an agent that specifically inhibits the lactation modification of XLF protein in the preparation of drugs to improve the chemosensitivity of colorectal cancer or reverse tumor chemosensitivity, characterized in that: The tumor is colorectal cancer, and the preparation is a polypeptide with the sequence HLYVSPWGGQRPQLSKVKRKKPRG.

2. The application of a reagent for detecting the expression level of lactation modification of XLF protein in the preparation of a kit for predicting the efficacy of tumor chemotherapy, characterized in that: The lactation modification site is lysine 288 of the XLF protein; the tumor is colorectal cancer.

3. The application according to claim 2, characterized in that: The level of lactation modification of the XLF protein is regulated by lysine acetyltransferase GCN5.

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