A reagent for inhibiting Acetyl-CoA or acetyltransferase GCN5 and its application

Abstract

This invention discloses a reagent for inhibiting Acetyl-CoA or GCN5 acetyltransferase and its application, belonging to the field of biomedical technology; a drug comprising a reagent capable of inhibiting Acetyl-CoA synthesis or GCN5 inhibition; tumor stem cells interact with T cells, and T cells can obtain a large amount of Acetyl-CoA from tumor stem cells; Acetyl-CoA, as an acetylation substrate, can acetylate Blimp-1 under the action of GCN5 acetyltransferase, downregulating Blimp-1 expression, thereby inhibiting TRM cell differentiation; by targeting and inhibiting Acetyl-CoA synthesis and GCN5 inhibition, Blimp-1 expression is upregulated, TRM cell differentiation is maintained, and the body's anti-tumor ability is improved; the Acetyl-CoA and GCN5 provided by this invention provide new targets for the treatment of NSCLC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a reagent for inhibiting Acetyl-CoA or acetyltransferase GCN5 and its application. Background Technology

[0002] Lung cancer has an extremely high incidence rate in my country and is a leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) is the main type of lung cancer, accounting for approximately 80%-85% of all cases. Currently, surgical treatment is the primary approach for stage I-II NSCLC patients; for patients with unresectable locally advanced NSCLC, radiotherapy combined with immunotherapy has defined a new standard of care. Although various immunotherapy methods exist, immunotherapy is not without its limitations; its response rate is limited, and it can cause organ-specific inflammatory side effects, even multiple organ failure. Therefore, new therapeutic targets and treatment regimens are still urgently needed.

[0003] Tumor immunotherapy is based on the presence of immune cells in the tumor tissue microenvironment (TME). The TME is a complex system primarily composed of immune cells, tumor-associated fibroblasts, nearby stromal tissue, microvessels, and various cytokines and chemokines, supporting tumor growth. Tumors develop under conditions of immunosuppression or exhaustion, indicating impaired immune cell function within the TME. Studies have found that immune cells infiltrating tumor tissue can exert both anti-tumor and pro-tumor effects. Immune cells in the TME, including macrophages, bone marrow-derived suppressor cells, natural killer cells, and adaptive immune cells, may contribute to creating an immunosuppressive environment to protect tumor cells from damage. For example, CD8+ cells chronically exposed to an inflammatory environment... + When TILs lose their effector function completely or partially, they become exhausted, and the dysfunction 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 Tm cell differentiation; at the same time, we investigated the clinical translational value of Acetyl-CoA or related acetyltransferases in the treatment of NSCLC, providing new technologies for the treatment of NSCLC. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a reagent for inhibiting Acetyl-CoA or acetyltransferase GCN5 and its application.

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

[0007] Preferably, the reagent includes one or more of ACLY inhibitors, ACSS2 inhibitors, ACLY shRNA, ACSS2 shRNA, and 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 above-mentioned agents that inhibit Acetyl-CoA synthesis or can inhibit GCN5 expression are used to promote Trm cell differentiation.

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

[0013] Preferably, the reagent includes one or more of ACLY inhibitors, ACSS2 inhibitors, ACLY shRNA, ACSS2 shRNA, and 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] The beneficial effects of this invention are:

[0016] The Acetyl-CoA and GCN5 of this invention provide new targets for the treatment of NSCLC. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0018] Figure 1 China A is a comprehensive database of gene expression in non-small cell lung cancer tissues from clinical patients, analyzing CSCs and CD8. + The correlation between Trm cells; B represents the relationship between CSCs / non-CSCs and CD8.+ After T cell co-culture, CD8 was detected by flow cytometry. + Trm cell differentiation status.

[0019] Figure 2 A represents the supernatant of CSCs / non-CSCs and CD8. + After T cell co-culture, the kit was used to detect CD8. + Figure T shows the Acetyl-CoA content; B shows the CD8 concentration detected by flow cytometry after inhibiting Acetyl-CoA with an inhibitor. + Diagram of Trm cell differentiation.

[0020] Figure 3 In the middle, A represents the effect of Western blot detection on CD8. + A) P) P) P) P) P) P) P) P) P) P) P) P) P) P)" ..."——Plasma structure of Blimp-1" P"" P) P) P) P)"""""" -—Plasma structure of Blimp-1" P" + During T cell culture, acetate was added, and the protein expression and acetylation levels of Blimp-1 were detected by immunoprecipitation.

[0021] Figure 4 A) Using tool cells, overexpression plasmids of five types of acetyltransferases (CBP, P300, GCN5, PCAF, and TIP60) were transfected, and the protein expression and acetylation levels of Blimp-1 were detected by immunoprecipitation. B) CSCs and CD8 + T cell co-culture, immunoprecipitation detection of CD8 + The binding of Blimp-1 to GCN5, PCAF, and TIP60 in Trm cells.

[0022] Figure 5 A shows a growth curve based on a humanized mouse model, where Acetyl-CoA synthesis in CSCs was inhibited using ACLY and ACSS2 lentiviral shRNA, and tumor growth was monitored. B shows a flow cytometry analysis of CD8+ in tumor tissue after Acetyl-CoA synthesis in CSCs was inhibited using ACLY and ACSS2 lentiviral shRNA, also based on a humanized mouse model. + Trm cell infiltration; C is a growth curve based on a humanized mouse model, monitoring tumor growth after inhibiting GCN5 in T cells with GCN5 lentiviral shRNA; D is a flow cytometry analysis of CD8 in tumor tissue based on a humanized mouse model, after inhibiting GCN5 in T cells with GCN5 lentiviral shRNA. + Trm cell infiltration status. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 purchased from ATCC; CD8+ cells from healthy individuals + T cells were obtained from 30 recruited healthy volunteers in a 1:1 male-to-female ratio. Peripheral blood samples were obtained from the volunteers with informed consent, and CD8+ cells were then sorted. + T cells. The experimental method has been approved by the Ethics Committee of Soochow University.

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

[0028] 1.2 Experimental Procedure

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

[0030] (1) A549 uses 5×10 3 The cells were seeded at a density of 0.5 ml in 6-well plates with low adsorption.

[0031] (2) Serum-free culture of DM EM / F12 supplemented with 1×B27, insulin (4 μg / ml), EGF (20 ng / ml), and FGF (20 ng / ml) was performed at 37°C.

[0032] 1.2.2CD8 + T cell sorting

[0033] (1) Reviving frozen PBMCs.

[0034] (2) Use T cell sorting buffer (PBS + 2% FBS + 1mM EDTA) at 1×10 7 The cells were resuspended at a ratio of 1 / 100 μl and transferred to sterile sorting tubes.

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

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

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

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

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

[0040] Using a Transwell chamber non-contact co-culture method, CSCs / non-CSCs were seeded in the lower chamber, and T cells were seeded in the upper chamber, with CSCs and non-CSCs co-cultured with CD8. + 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 co-culturing T cells for 24 hours, CD3 / CD28 beads and TGF-β (10 ng / ml) were added to stimulate and induce them for 3 days.

[0043] 1.2.5 Flow cytometry detection of cell differentiation

[0044] (1) Take differentiated 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 stop staining, centrifuge at 1700 rpm for 6 minutes and discard the supernatant.

[0048] (5) Add 200 μL of PBS to resuspend the cells, transfer them to a flow cytometer and run them on the flow cytometer.

[0049] 1.3 Experimental Results

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

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

[0052] 1. Experimental materials and reagents

[0053] (1) Cells: Same as control group 1.

[0054] (2) Reagents: ACLY inhibitor (AbMole), ACSS2 inhibitor (Selleck), Acetyl-CoA detection kit (Abcam), the rest are the same as in comparative example 1.

[0055] 2. Experimental Procedure

[0056] Cells were pretreated with inhibitors.

[0057] (1) Dissolve ACLY and ACSS2 inhibitors in DMSO, aliquot and store at -80℃.

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

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

[0060] 3. Experimental Results

[0061] Building CSCs / non-CSCs with CD8 + A co-culture platform for T cells revealed that during co-culture with CSCs, CD8... +T cells can acquire large amounts of Acetyl-CoA; simultaneously, it was found that inhibiting Acetyl-CoA synthesis with ACLY and AC SS2 inhibitors can effectively block CSCs' inhibition of CD8. + Trm cell differentiation, results are shown in Figure 2 .

[0062] Example 2 explores the effect of Acetyl-CoA on Blimp-1

[0063] 1. Experimental materials and reagents

[0064] (1) Cells: Same as control group 1.

[0065] (2) Reagents: Anti-Acetyl-Lysine antibody (Abclonal), Blimp-1, Hobit, RUNX1, RUNX3 antibody (Invitrogen), the rest are the same as above.

[0066] 2. Experimental Procedure

[0067] Western blot analysis of protein expression

[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 containing PMSF (approximately 100 μl / 1×10⁻⁶). 6 Cells were lysed on ice for 30 minutes (1 cell).

[0071] ③ Centrifuge at 4℃ and 12000rpm for 15 minutes.

[0072] ④ Draw the supernatant into another EP tube, add 1 / 4 volume of 5×loading and mix thoroughly by blowing.

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

[0074] (2) Western Blot:

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

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

[0077] ③ Transfer membrane:

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

[0079] 2) Clamp the transfer clamps in the order of black adhesive and white film (protein adhesive near the black cathode carbon plate, PVDF film near the white transparent anode carbon plate), and make sure there are no air bubbles between the adhesive and the film.

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

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

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

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

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

[0085] Immunoprecipitation technology

[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] ② Add an appropriate amount of IP cell lysis buffer containing PMSF (approximately 100 μl / 1×10⁻⁶). 6 Cells were lysed on ice for 30 minutes (1 cell).

[0089] ③ Centrifuge at 4℃ and 12000rpm for 15 minutes.

[0090] ④ Transfer the supernatant to 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] ⑤ Take ten times the volume of antibody Protein A / G beads into an EP tube, use a magnetic rack to adsorb the beads and discard the protective solution, wash the beads twice with IP lysis buffer, and then add them to the protein sample from step ④. Vortex at 4°C for 3-5 hours.

[0092] ⑥ Adsorb the magnetic beads onto the magnetic rack, discard the clear liquid, add IP washing buffer and wash 3-4 times, then wash once with high-salt solution.

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

[0094] 3 Experimental Results

[0095] Building CSCs / non-CSCs with CD8 + A co-culture platform for T cells revealed that co-culturing CD8 cells with CSCs improved their efficacy. + The level of Blimp-1 protein in T cells was significantly reduced, while Hobit, Runx1, and Runx3 showed no significant changes; after co-culturing with CSCs, CD8 + The acetylation level of Blimp-1 in T cells was significantly upregulated, accompanied by a decrease in Blimp-1 protein levels; when the Acetyl-CoA precursor acetate was added, CD8+ from healthy individuals... + Increased Blimp-1 acetylation levels in T cells were accompanied by downregulation of Blimp-1 protein levels. Results are shown below. Figure 3 .

[0096] Example 3: Investigating the key acetyltransferase that promotes Blimp-1 acetylation 1. Experimental materials and reagents

[0097] (1) Cells: HEK293T cell line was purchased from ATCC, and the rest were the same as in control group 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 Procedure

[0100] PEI transfection

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

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

[0103] (3) Transfer the transfection reagent into 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 transformed.

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

[0106] 3 Experimental Results

[0107] Based on the above experimental results, it was found that after transfecting 293T tools cells with CBP, P300, GCN5, PCAF, and TIP60, the acetylation level of Blimp-1 was significantly upregulated, accompanied by a downregulation of Blimp-1 protein levels; constructing CSCs / non-CSCs and CD8... + T cell co-culture platform, CD8 co-cultured with CSCs + In Trm cells, only GCN5 and Blimp-1 bind directly. Results are shown below. Figure 4 .

[0108] Comparative Example 2

[0109] 1. Experimental materials and reagents:

[0110] (1) Cells / Tissues: Lung tissue and blood samples from 20 recruited NSCLC patients (male-female ratio 1:1, mean age 65±10 years) were obtained with informed consent. These samples were used to construct a PDO model, establish a humanized mouse model, and conduct translational research. CD8+ samples from healthy individuals were also collected. + T cell source same as control ratio 1.

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

[0112] 2. Experimental Procedure:

[0113] CSCs single-cell sorting of NSCLC PDOs

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

[0115] (2) Add CD133 flow cytometry antibody and stain;

[0116] (3) The CD133 positive CSCs were obtained by using a flow cytometer.

[0117] Tumor tissue growth monitoring:

[0118] The three-dimensional volume of tumor tissue was measured periodically using vernier calipers.

[0119] Lentiviral transfection:

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

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

[0122] (3) After 48 hours, collect the supernatant, centrifuge to obtain virus particles, and store at -80℃.

[0123] (4) Lentiviral 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 was the same as that in Comparative Example 1.

[0126] 3 Experimental Results

[0127] The results are as follows Figure 5 .

[0128] Example 4: Constructing a humanized mouse model to investigate the protective effects of inhibiting Acetyl-CoA and GCN5 on NSCLC.

[0129] 1. Experimental materials and reagents

[0130] (1) Cells / Tissues: Lung tissue and blood samples from 20 recruited NSCLC patients (male-female ratio 1:1, mean age 65±10 years) were obtained with informed consent. These samples were used to construct a PDO model, establish a humanized mouse model, and conduct translational research. CD8+ samples from healthy individuals were also collected. + T cell source same as control ratio 1.

[0131] (2) CD133 flow cytometry antibody (Invitrogen), GCN5 / ACLY / ASSC2 shRNA (Anzhen Biotechnology), 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 Procedure

[0136] CSCs single-cell sorting of NSCLC PDOs

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

[0138] (2) Add CD133 flow cytometry antibody and stain;

[0139] (3) The CD133 positive CSCs were obtained by using a flow cytometer.

[0140] Tumor tissue growth monitoring

[0141] The three-dimensional volume of tumor tissue was measured periodically using vernier calipers.

[0142] Lentiviral transfection

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

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

[0145] (3) After 48 hours, collect the supernatant, centrifuge to obtain virus particles, and store at -80℃.

[0146] (4) Lentiviral 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 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 was the same as that in Comparative Example 1.

[0149] 3 Experimental Results

[0150] A humanized mouse model was constructed to block the synthesis of CSCsAcetyl-CoA and downregulate CD8. + The expression of acetyltransferase GCN5 in T cells can effectively block the effect of CSCs on CD8. + The study investigated the effects of tumor regeneration (TRM) cell differentiation on tumor cells (CSCs). Furthermore, by monitoring tumor growth over 30 days, it was found that inhibition of Acetyl-CoA synthesis and CD8+ in CSCs was observed. +T cell GCN5 expression inhibits tumor growth. Results are shown below. Figure 5 .

[0151] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0152] The foregoing has shown and described 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. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A drug, characterized in that, Including agents that inhibit Blimp-1 acetylation by inhibiting Acetyl-CoA synthesis or inhibiting GCN5 expression to promote TRM cell differentiation; The reagents include one or more of ACLY shRNA, ACSS2 shRNA, and GCN5 shRNA; 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.

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

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

4. The application of the reagent according to any one of claims 1 to 3, which promotes Trm cell differentiation by inhibiting Acetyl-CoA synthesis or inhibiting GCN5 expression, in the preparation of drugs for treating non-small cell lung cancer; The reagents include one or more of ACLYshRNA, ACSS2 shRNA, and GCN5 shRNA; 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.

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