Reagent for inhibiting Acetyl-CoA or acetylase GCN5 and application thereof

Abstract

The invention discloses a reagent for inhibiting Acetyl-CoA or acetylase GCN5 and application of the reagent, and belongs to the technical field of biological medicine. A medicament comprising an agent capable of inhibiting the synthesis of Acetyl-CoA or capable of inhibiting GCN5; the tumor stem cells and the T cells interact, and the T cells can obtain a large amount of Acetyl-CoA from the tumor stem cells; according to the present invention, the Acetyl-CoA is adopted as the acetylation substrate, and under the action of the acetylase GCN5, the Blimp-1 can be acetylated, and the expression of the Blimp-1 can be down-regulated so as to inhibit the Trm cell differentiation; through targeted inhibition of synthesis of Acetyl-CoA and inhibition of GCN5, expression of Blimp-1 is up-regulated, Trm cell differentiation is maintained, and the anti-tumor ability of an organism is improved; the Acetyl-CoA and the GCN5 provided by the invention provide a new target spot for the treatment of the NSCLC (Non-Small Cells Leukemia).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a reagent for inhibiting Acetyl-CoA or acetylase GCN5 and an application thereof. Background Art

[0002] Lung cancer has an extremely high incidence in my country and is also the leading cause of cancer-related deaths. Non-small cell lung cancer is the main type of lung cancer, accounting for about 80%-85% of the total number of patients. At present, the clinical treatment for patients with stage I-II NS CLC is mainly surgical treatment; for patients with locally advanced disease that cannot be resected, chemoradiotherapy combined with immunotherapy defines a new treatment standard. Although there are currently a variety of immunotherapy methods, immunotherapy is not perfect, its response rate is limited, and it can cause organ-specific inflammatory side effects and even multiple organ failure. Therefore, new therapeutic targets and treatment options are still urgently needed to be discovered.

[0003] Tumor immunotherapy is based on the presence of immune cells in the tumor tissue microenvironment. The tumor microenvironment is a complex integrated system mainly composed of immune cells, tumor-associated fibroblasts, nearby stromal tissue, microvessels, and various cytokines and chemokines to support tumor growth. Tumors occur under conditions of immunosuppression or exhaustion, which means that the function of immune cells in the TME is impaired. Studies have found that immune cells infiltrating tumor tissues can have both anti-tumor and pro-tumor effects. Immune cells in the TME, including macrophages, myeloid-derived suppressor cells, natural killer cells, and adaptive immune cells, may help form an immunosuppressive environment to protect tumor cells from damage. For example, CD8 + The TIL effector function is completely or partially lost, presenting a state of exhaustion, and the disability of T cells will eventually lead to immune escape.

[0004] Therefore, based on the interaction between tumor stem cells and T cells in the tumor microenvironment, we explored the role and mechanism of Acetyl-CoA in CSCs or related acetyltransferases in T cells on Trm cell differentiation; at the same time, we targeted Acetyl-CoA or related acetyltransferases to explore their clinical translational value in the treatment of NSCLC and provide new technologies for the treatment of NSCLC. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a reagent for inhibiting Acetyl-CoA or acetylase GCN5 and its application.

[0006] A drug comprising an agent capable of inhibiting the synthesis of Acetyl-CoA or capable of inhibiting the expression of GCN5.

[0007] Preferably, the agent comprises one or more of an ACLY inhibitor, an ACSS2 inhibitor, an ACLY shRNA, an ACSS2 shRNA and a GCN5 shRNA.

[0008] Preferably, the sequence of the ACLY shRNA is SEQ ID NO: 1; the sequence of the GCN5 shRNA is SEQ ID NO: 2; and the sequence of the ACSS2 shRNA is SEQ ID NO: 3.

[0009] Preferably, the Acetyl-CoA is derived from tumor stem cells.

[0010] Preferably, the GCN5 is derived from T cells.

[0011] The use of the above-mentioned reagents capable of inhibiting Acetyl-CoA synthesis or inhibiting GCN5 expression in promoting Trm cell differentiation.

[0012] The above reagents capable of inhibiting the synthesis of Acetyl-CoA or inhibiting the expression of GCN5 are used in the preparation of drugs for treating non-small cell lung cancer.

[0013] Preferably, the agent comprises one or more of an ACLY inhibitor, an ACSS2 inhibitor, an ACLY shRNA, an ACSS2 shRNA and a GCN5 shRNA.

[0014] Preferably, the sequence of the ACLY shRNA is SEQ ID NO: 1; the sequence of the GCN5 shRNA is SEQ ID NO: 2; and the sequence of the ACSS2 shRNA is SEQ ID NO: 3.

[0015] Beneficial effects of the present invention:

[0016] The Acetyl-CoA and GCN5 of the present invention provide new targets for treating NSCLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 A in the figure is based on the comprehensive gene expression database of non-small cell lung cancer tissues of clinical patients, analyzing the relationship between CSCs and CD8 + The correlation between Trm cells; B is CSCs / non-CSCs and CD8+ After co-culture of T cells, flow cytometry was used to detect CD8 + Trm cell differentiation.

[0019] Figure 2 A in the figure shows the supernatant of CSCs / non-CSCs and CD8 + After T cell co-culture, the kit detects CD8 + The content of Acetyl-CoA in T; B is the flow cytometry detection of CD8 after Acetyl-CoA was inhibited by inhibitor + Diagram of Trm cell differentiation.

[0020] Figure 3 A in the middle is Western blot detection of CD8 + The protein expression level of Blimp-1, Hobit, RUNX1 and RUNX3, key transcription factors for Trm cell differentiation; B is the protein expression level and acetylation level of Blimp-1 detected by immunoprecipitation; C is the protein expression level and acetylation level of healthy human CD8 + Acetate was added while T cells were cultured, and the protein expression level and acetylation level of Blimp-1 were detected by immunoprecipitation.

[0021] Figure 4 A shows the use of tool cells to transfect overexpression plasmids of five types of acetylases, namely CBP, P300, GCN5, PCAF, and TIP60, and immunoprecipitation to detect the protein expression level and acetylation level of Blimp-1; B shows the relationship between CSCs and CD8 + T cell co-culture and immunoprecipitation to detect CD8 + The binding of Blimp-1 with GCN5, PCAF and TIP60 in Trm cells.

[0022] Figure 5 A is a growth curve chart based on a humanized mouse model, in which ACLY and ACSS2 lentiviral shRNAs were used to inhibit the synthesis of Acetyl-CoA in CSCs, and the growth of the tumor was monitored; B is a flow cytometry chart based on a humanized mouse model, in which ACLY and ACSS2 lentiviral shRNAs were used to inhibit the synthesis of Acetyl-CoA in CSCs, and the CD8 + Trm cell infiltration; C is a growth curve chart based on a humanized mouse model, after GCN5 lentiviral shRNA was used to inhibit GCN5 in T cells, monitoring tumor growth; D is a flow cytometry chart based on a humanized mouse model, after GCN5 lentiviral shRNA was used to inhibit GCN5 in T cells, detecting CD8 + Trm cell infiltration. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Comparative Example 1

[0025] 1.1 Experimental Materials and Reagents

[0026] (1) Cells: A549 cell line was purchased from ATCC; healthy CD8 + T cells were obtained from 30 healthy volunteers, with a male-female ratio of 1:1. Peripheral blood samples were obtained under informed conditions and CD8 + T cells. The experimental methods were approved by the Ethics Committee of Soochow University.

[0027] (2) Main reagents: EasySepTM Human CD8 + T cell isolation kit (Stemcell), CD3 / CD28 beads (Thermo Fisher Scientific), anti-Human CD103, anti-Human CD8 flow cytometry antibodies (Biolegend), DMEM / F12 medium (Corning).

[0028] 1.2 Experimental process

[0029] 1.2.1 Enrichment of non-small cell lung cancer stem cells

[0030] (1) A549 with 5×10 3 The cells were seeded at a density of 1.54 g / ml in a 6-well plate with low adsorption.

[0031] (2) The cells were cultured at 37°C in DM EM / F12 serum-free medium supplemented with 1×B27, insulin (4 μg / ml), EGF (20 ng / ml), and FGF (20 ng / ml).

[0032] 1.2.2CD8 + T cell sorting

[0033] (1) Thaw cryopreserved PBMCs.

[0034] (2) Use T cell sorting buffer (PBS + 2% FBS + 1mM EDTA) at 1×10 7 Resuspend the cells at a ratio of 1:1 / 100 μl and transfer to a sterile sorting tube.

[0035] (3) 1×10 7 Add the Isolation Cocktail in the Isolation Kit at a ratio of 1:1 / 10 μl and incubate at room temperature for 5 minutes.

[0036] (4) Vortex RapidSpheres, 1×10 7 Add to cells at a ratio of 1:1 / 10 μl and mix well.

[0037] (5) Add sorting buffer to 2.5 ml and transfer the sorting tube to the magnetic rack for adsorption for 5 minutes.

[0038] (6) Aspirate the cell fluid and centrifuge it. Resuspend it in 1640 complete medium and use it for downstream experiments.

[0039] 1.2.3 CSCs / non-CSCs and CD8 + T cell co-culture system

[0040] Using the non-contact co-culture method of Transwell chamber, CSCs / non-CSCs were seeded in the lower chamber, and T cells were seeded in the upper chamber. + T cells were co-cultured at a 1:1 ratio for 24 hours.

[0041] 1.2.4CD8 + T cell stimulation, activation and induction

[0042] CSCs / non-CSCs and CD8 + After T cells were co-cultured for 24 hours, CD3 / CD28 beads and TGF-β (10 ng / ml) were added for stimulation and induction for 3 days.

[0043] 1.2.5 Flow cytometry to detect cell differentiation

[0044] (1) Take differentiated and mature CD8 + T cells were centrifuged at 1700 rpm for 6 minutes and the supernatant was discarded.

[0045] (2) Resuspend the cells in 100 μl of PBS containing 2% FBS.

[0046] (3) Add 0.5 μl each of anti-Human CD8 and anti-Human CD103 flow cytometry antibodies and stain on ice for 30 minutes.

[0047] (4) Add about 400 μl of PBS to terminate staining, centrifuge at 1700 rpm for 6 minutes, and discard the supernatant.

[0048] (5) Add 200ul PBS to resuspend the cells, transfer to a flow cytometry tube and load onto the instrument.

[0049] 1.3 Experimental Results

[0050] Construction of CSCs / non-CSCs and CD8 + T cell co-culture platform, compared with the non-CSCs control group, CD8 + After co-culture of T cells and CSCs, CD8 + The reduced differentiation ability of Trm cells indicates that CSCs inhibit CD8 + The results of Trm cell differentiation are shown in Figure 1 .

[0051] Example 1 Detection of whether CSCs-derived Acetyl-CoA inhibits CD8 + Trm cell differentiation

[0052] 1. Experimental materials and reagents

[0053] (1) Cells: Same as Comparative Example 1.

[0054] (2) Reagents: ACLY inhibitor (AbMole), ACSS2 inhibitor (Selleck), Acetyl-Co A detection kit (Abcam), and the rest are the same as in Comparative Example 1.

[0055] 2. Experimental process

[0056] Cells were pre-treated with inhibitors.

[0057] (1) Dissolve ACLY and ACSS2 inhibitors in DMSO and store in aliquots at -80°C.

[0058] (2) CSCs were pre-treated with ACLY (40uM) and ACSS2 (10uM) inhibitors, and the same volume of solvent DMSO was added to the control group for 24 hours.

[0059] Flow cytometry detection of CD8 + The experimental method for Trm cell differentiation is the same as that of Comparative Example 1.

[0060] 3. Experimental results

[0061] Construction of CSCs / non-CSCs and CD8 + The co-culture platform of T cells found that during co-culture with CSCs, CD8 +T cells can obtain a large amount of Acetyl-CoA; at the same time, it was found that the use of ACLY and ACSS2 inhibitors to inhibit the synthesis of Acetyl-CoA can effectively block the effect of CSCs on CD8 + The results of Trm cell differentiation are shown in Figure 2 .

[0062] Example 2: Effect of Acetyl-CoA on Blimp-1

[0063] 1 Experimental materials and reagents

[0064] (1) Cells: Same as Comparative Example 1.

[0065] (2) Reagents: anti-Acetyl-Lysine antibody (Abclonal), Blimp-1, Hobit, RUNX1, RU NX3 antibodies (Invitrogen), and the rest are the same as above.

[0066] 2 Experimental process

[0067] Protein expression detected by Western Blot

[0068] (1) Sample preparation:

[0069] ① Collect differentiated and mature CD8 + T cells were centrifuged at 1700 rpm for 6 minutes and the supernatant was discarded.

[0070] ② Add an appropriate amount of RIPA cell lysis buffer (about 100 μl / 1×10 6 Cells were lysed on ice for 30 min.

[0071] ③Centrifuge at 4°C, 12000rpm for 15 minutes.

[0072] ④ Pipette the supernatant into another EP tube, add 1 / 4 volume of 5× loading medium and mix well by pipetting.

[0073] ⑤ Denature the protein in a 100℃ metal bath for 10 minutes.

[0074] (2) Western Blot:

[0075] ① Gel preparation: Select a separation gel of appropriate concentration according to the molecular weight of the target protein and prepare it according to the instructions.

[0076] ② Gel running: Add an appropriate amount of protein sample (usually no more than 50μl for 10-well gel and no more than 25μl for 15-well gel), and run the entire process at a voltage of 200V. The electrophoresis time can be adjusted according to experimental requirements.

[0077] ③Transfer:

[0078] 1) Activate the PVDF membrane with anhydrous methanol in advance.

[0079] 2) Clamp the transfer clamp in the order of black gel and white film (protein gel is close to the black cathode carbon plate, PVDF film is close to the white transparent anode carbon plate), and make sure there are no bubbles between the gel and the film.

[0080] 3) Place the transfer clip in the transfer tank, pour in the pre-cooled transfer solution, and transfer in an ice bath. (The transfer current and time can be adjusted according to the size of the protein).

[0081] ④ Blocking: Prepare blocking solution (5% skim milk or 5% BSA) with PBST and block at room temperature for 1 hour.

[0082] ⑤ Primary antibody incubation: After blocking, wash the membrane three times with PBST, 10 minutes each time, and then add the diluted primary antibody and incubate overnight at 4°C.

[0083] ⑥ Secondary antibody incubation: After the primary antibody incubation, wash the membrane three times with PBST, 10 minutes each time. Dilute the secondary antibody with PBST (1:8000) and incubate at room temperature for 1 hour.

[0084] ⑦Exposure: Prepare an appropriate amount of ECL luminescent liquid (liquid A: liquid B = 1:1) and expose.

[0085] Immunoprecipitation technique

[0086] (1) Sample preparation

[0087] ① Collect differentiated and mature CD8 + T cells were centrifuged at 1700 rpm for 6 minutes and the supernatant was discarded.

[0088] ②Add an appropriate amount of IP cell lysis buffer with PMSF added (about 100 μl / 1×10 6 Cells were lysed on ice for 30 min.

[0089] ③Centrifuge at 4°C, 12000rpm for 15 minutes.

[0090] ④ Pipette the supernatant into another EP tube, add an appropriate amount of IP antibody (the amount of different antibodies can be referred to the instructions), and vortex overnight at 4°C.

[0091] ⑤Put ten times the volume of the antibody in Protein A / G beads into an EP tube, adsorb the beads on a magnetic stand and discard the protective solution, wash the beads twice with IP lysis buffer, then add them to the protein sample in step ④ and vortex at 4℃ for 3-5 hours.

[0092] ⑥ Adsorb the magnetic beads on the magnetic stand, discard the supernatant, add IP washing buffer and wash 3-4 times, and wash once with high salt solution.

[0093] ⑦ Add 30 μl of 1× loading solution diluted with lysis buffer to the magnetic beads and place in a 100℃ metal bath for 10 minutes to denature the protein. (2) Western Blot experiment is the same as above.

[0094] 3 Experimental results

[0095] Construction of CSCs / non-CSCs and CD8 + T cell co-culture platform, found that CD8 + The level of Blimp-1 protein in T cells was significantly decreased, while there was no significant change in Hobit, Runx1, and Runx3. After co-culture with CSCs, CD8 + The acetylation level of Blimp-1 in T cells was significantly upregulated, accompanied by a decrease in the level of Blimp-1 protein. When the Acetyl-CoA precursor acetate was added, the CD8 + The acetylation level of Blimp-1 in T cells increased, accompanied by the downregulation of Blimp-1 protein level. Figure 3 .

[0096] Example 3: Exploring the Key Acetylase 1 that Promotes Blimp-1 Acetylation Experimental Materials and Reagents

[0097] (1) Cells: HEK293T cell line was purchased from ATCC, and the rest was the same as in Comparative Example 1.

[0098] (2) Reagents: DDDDK-Tag antibody (Abclonal), anti-Myc-Tag antibody (Abmart), HA-Tag antibody (Abclonal), anti-GCN5 antibody (Abclonal), anti-PCAF antibody (Abclonal), anti-Tip60 antibody (Abclonal), and the rest are the same as above.

[0099] 2 Experimental process

[0100] PEI transfection

[0101] (1) Prepare two 1.5 ml sterile EP tubes and add an appropriate amount of Opti-MEM medium to each tube (the amount added depends on the number of cells to be transfected, generally 50 μl for a 12-well plate).

[0102] (2) Add the plasmid to be transfected and three times the amount of transfection reagent (the plasmid concentration is related to the number of cells transfected, generally 2 μg is transfected in a 12-well plate) into two EP tubes, mix well and let stand for 5 minutes.

[0103] (3) Transfer the transfection reagent to the plasmid, mix well, and let stand for 15 minutes.

[0104] (4) Dilute the mixture with complete culture medium and add it to the cells to be transfected.

[0105] The experimental method for detecting Blimp-1 acetylation and its binding to acetylase by immunoprecipitation technique is the same as that in Example 2.

[0106] 3 Experimental results

[0107] According to the above experimental results, after the tool cells 293T were transfected with CBP, P300, GCN5, PCAF, and TIP60, the acetylation level of Blimp-1 was significantly upregulated, accompanied by the downregulation of Blimp-1 protein level; + Co-culture platform of T cells, CD8 + Only GCN5 and Blimp-1 in Trm cells directly bind. Figure 4 .

[0108] Comparative Example 2

[0109] 1 Experimental materials and reagents:

[0110] (1) Cells / tissues: Lung tissues and blood samples of NSCLC patients were obtained from 20 NSCLC patients recruited, with a male-to-female ratio of 1:1 and an average age of 65±10 years. With their informed consent, tumor tissue and blood samples were obtained to construct PDO models, establish humanized mouse models, and conduct translational research; healthy CD8 + The source of T cells is the same as that of Comparative Example 1.

[0111] (2) CD133 flow cytometry antibody (Invitrogen), the rest is the same as above.

[0112] 2 Experimental process:

[0113] Isolation of single-cell CSCs from NSCLC PDOs

[0114] (1) PDO was digested with collagenase IV for 2 h, followed by isolation of single cell suspension;

[0115] (2) adding CD133 flow cytometry antibody and staining;

[0116] (3) Using a flow cytometer to sort, CD133-positive CSCs were obtained.

[0117] Tumor tissue growth monitoring:

[0118] The three-dimensional volume of tumor tissue was measured regularly using a vernier caliper.

[0119] Lentiviral transfection:

[0120] (1) Cultivate 293T cells until the cells are in good condition and the density reaches 30%-50%;

[0121] (2) Co-transfection of envelope plasmid (psPAX2, pMD2.G).

[0122] (3) After 48 hours, the supernatant was collected, centrifuged to obtain viral particles, and stored at -80°C.

[0123] (4) Lentivirus infection of CSCs: those transfected with control shRNA served as the control group;

[0124] (5) Lentiviral transfection of CD8 + T cells: those transfected with control shRNA served as the control group.

[0125] Flow cytometry detection of CD8 + The experimental method for Trm cell differentiation is the same as that of Comparative Example 1.

[0126] 3 Experimental results

[0127] The results are as follows Figure 5 .

[0128] Example 4 Construction of a humanized mouse model to explore the protective effect of inhibiting Acetyl-CoA and GCN5 on NSCLC

[0129] 1 Experimental materials and reagents

[0130] (1) Cells / tissues: Lung tissues and blood samples of NSCLC patients were obtained from 20 NSCLC patients recruited, with a male-to-female ratio of 1:1 and an average age of 65±10 years. With their informed consent, tumor tissue and blood samples were obtained to construct PDO models, establish humanized mouse models, and conduct translational research; healthy CD8 + The source of T cells is the same as that of Comparative Example 1.

[0131] (2) CD133 flow cytometry antibody (Invitrogen), GCN5 / ACLY / ASSC2 shRNA (Anzhen Biotechnology), and the rest are the same as above.

[0132] The sequence of ACLY shRNA is CGTGAGAGCAATTCGAGATTAT.

[0133] The sequence of GCN5 shRNA is CGATGTTCGAGCTCTCAAAGAT.

[0134] The sequence of ASSC2 shRNA is GATGCTGCATTGTGGTCAAT.

[0135] 2 Experimental process

[0136] Isolation of single-cell CSCs from NSCLC PDOs

[0137] (1) PDO was digested with collagenase IV for 2 h, followed by isolation of single cell suspension;

[0138] (2) adding CD133 flow cytometry antibody and staining;

[0139] (3) Using a flow cytometer to sort, CD133-positive CSCs were obtained.

[0140] Tumor tissue growth monitoring

[0141] The three-dimensional volume of tumor tissue was measured regularly using a vernier caliper.

[0142] Lentiviral transfection

[0143] (1) Cultivate 293T cells until the cells are in good condition and the density reaches 30%-50%;

[0144] (2) Co-transfection of shRNAs (shACLY, shACSS2, shGCN5, control shRNA) and envelope plasmids (psPAX2, pMD2.G).

[0145] (3) After 48 hours, the supernatant was collected, centrifuged to obtain viral particles, and stored at -80°C.

[0146] (4) Lentivirus infection of CSCs: CSCs were transfected with shACLY / shACSS2 to block the synthesis of Acetyl-CoA in CSCs.

[0147] (5) Lentiviral transfection of CD8 + T cells: CD8 cells were transfected with shGCN5 + T cells downregulate the expression of GCN5 in T cells,

[0148] Flow cytometry detection of CD8 + The experimental method for Trm cell differentiation is the same as that of Comparative Example 1.

[0149] 3 Experimental results

[0150] Construction of a humanized mouse model to block the synthesis of Acetyl-CoA in CSCs and downregulate CD8 + The expression of acetylase GCN5 in T cells can effectively block CSCs from CD8 + The effects of TNF-α on Trm cell differentiation and the inhibition of Acetyl-CoA synthesis and CD8 in CSCs were also observed by monitoring tumor growth for 30 days. +The expression of GCN5 in T cells inhibited tumor growth. Figure 5 .

[0151] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0152] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A drug, characterized in that Included are agents capable of inhibiting Acetyl-CoA synthesis or capable of inhibiting GCN5 expression.

2. The drug according to claim 1, characterized in that The reagents include one or more of an ACLY inhibitor, an ACSS2 inhibitor, an ACLY shRNA, an ACSS2 shRNA, and a GCN5 shRNA.

3. The drug according to claim 2, characterized in that The sequence of the ACLY shRNA is SEQ ID NO: 1; the sequence of the GCN5 shRNA is SEQ ID NO: 2; and the sequence of the ACSS2 shRNA is SEQ ID NO:

3.

4. The drug according to claim 1, characterized in that The Acetyl-CoA is derived from tumor stem cells.

5. The drug according to claim 1, characterized in that The GCN5 is derived from T cells.

6. Use of the reagent capable of inhibiting Acetyl-CoA synthesis or GCN5 expression according to any one of claims 1 to 5 in promoting Trm cell differentiation.

7. Use of the reagent capable of inhibiting the synthesis of Acetyl-CoA or the expression of GCN5 according to any one of claims 1 to 5 in the preparation of a drug for treating non-small cell lung cancer.

8. Use of the reagent capable of inhibiting the synthesis of Acetyl-CoA or the expression of GCN5 according to claim 7 in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The reagents include one or more of an ACLY inhibitor, an ACSS2 inhibitor, an ACLY shRNA, an ACSS2 shRNA, and a GCN5 shRNA.

9. Use of the reagent capable of inhibiting the synthesis of Acetyl-CoA or the expression of GCN5 according to claim 8 in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The sequence of the ACLY shRNA is SEQ ID NO: 1; the sequence of the GCN5 shRNA is SEQ ID NO: 2; and the sequence of the ACSS2 shRNA is SEQ ID NO: 3.

Images