Application of baHD1 as a target in preparation of leukemia treatment drugs

Abstract

The application relates to application of BAHD1 as a target point in preparation of a leukemia treatment drug and belongs to the technical field of biological medicine. In order to solve the problems of high drug resistance and high recurrence of an acute leukemia treatment scheme, the application provides application of BAHD1 as a target point in preparation of a leukemia treatment drug. It is proved that BAHD1 is highly expressed in acute leukemia cells, and is used as a molecular marker for diagnosis of acute leukemia. It is proved that knocking out BAHD1 can significantly inhibit proliferation and self-renewal of the acute leukemia cells, promote apoptosis and differentiation of the acute leukemia cells, effectively inhibit proliferation of leukemia cells in the body, reduce tumor load and extramedullary infiltration, and significantly prolong the survival period. In addition, knocking out BAHD1 can significantly enhance the sensitivity of the acute leukemia cells to traditional chemotherapy drugs and targeted treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of BAHD1 as a target in the preparation of drugs for the treatment of leukemia. Background Technology

[0002] Leukemia is a type of malignant clonal disease of hematopoietic stem cells, classified into acute leukemia and chronic leukemia based on the stage of cell differentiation and maturation. Acute leukemia can be further divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) based on the affected cell type. The standard chemotherapy regimen for acute leukemia involves multiple rounds of high-intensity chemotherapy using anthracyclines combined with cytarabine or vincristine alkaloids combined with anthracyclines and cytarabine. Although most patients achieve complete remission after initial induction therapy, the vast majority eventually relapse due to the frequent emergence or evolution of drug-resistant clones. Therefore, identifying new therapeutic targets for acute leukemia and developing effective intervention strategies remain critical needs that urgently require addressing.

[0003] BAHD1, an important H3K27me3 reader, plays a crucial role in transcriptional repression and has been found to significantly inhibit multiple genes related to cell proliferation and survival, including IGF2. Its functions are remarkably diverse, participating not only in regulating basic growth and development processes but also playing a vital role in key stages such as erythrocyte maturation. However, research on BAHD1 in the field of cancer is relatively scarce, and its effects exhibit significant tissue heterogeneity: studies have shown that BAHD1 gene repression is associated with poor prognosis in lung cancer, and its expression is also generally downregulated in bladder cancer; conversely, high expression of BAHD1 is a risk factor for lymph node metastasis in breast cancer patients and predicts poor prognosis. Of particular note is that, despite the diverse functions of BAHD1 and its research in various cancers, its role in the development and progression of leukemia remains largely unknown. Summary of the Invention

[0004] To address the common problems of high drug resistance and high relapse rates in acute leukemia treatment regimens, this invention provides the application of BAHD1 as a target in the preparation of leukemia treatment drugs.

[0005] The technical solution of this invention:

[0006] Application of BAHD1 gene expression inhibitors in the preparation of drugs for the treatment of leukemia.

[0007] Furthermore, the BAHD1 gene expression inhibitor is an artificially designed small nucleic acid BAHD1 gene silencing agent.

[0008] Furthermore, the artificially designed small nucleic acid BAHD1 gene silencing agent is siRNA, and the sequence of the siRNA is shown in SEQ ID No:3.

[0009] Furthermore, the artificially designed small nucleic acid BAHD1 gene silencing agent is sgRNA, and the sequence of the sgRNA is shown in SEQ ID No:4 or SEQ ID No:5.

[0010] Furthermore, the artificially designed small nucleic acid BAHD1 gene silencing agent is shRNA, and the sequence of the shRNA is shown in SEQ ID No:6 or SEQ ID No:7.

[0011] Furthermore, the leukemia treatment drug is a combination of a man-made small nucleic acid BAHD1 gene silencing agent and cytarabine. The man-made small nucleic acid BAHD1 gene silencing agent is sgRNA, and the sequence of the sgRNA is shown in SEQ ID No:4 or SEQ ID No:5.

[0012] Furthermore, the leukemia treatment drug is a combination of a man-designed small nucleic acid BAHD1 gene silencing agent and a menin-KMT2A interaction inhibitor VTP50469. The man-designed small nucleic acid BAHD1 gene silencing agent is sgRNA, the sequence of which is shown in SEQ ID No:4 or SEQ ID No:5. The chemical formula of VTP50469 is C 32 H 47 FN6O4S, structural formula is

[0013] .

[0014] Furthermore, the leukemia treatment drug is a combination of a man-made small nucleic acid BAHD1 gene silencing agent and an IRE1 inhibitor STF-083010. The man-made small nucleic acid BAHD1 gene silencing agent is sgRNA, the sequence of which is shown in SEQ ID No:4 or SEQ ID No:5, and the chemical formula of STF-083010 is C 15 H 11 NO3S2, structural formula is

[0015] .

[0016] Furthermore, the leukemia treatment drug is a combination of a man-made small nucleic acid BAHD1 gene silencing agent and a PERK inhibitor GSK2606414. The man-made small nucleic acid BAHD1 gene silencing agent is sgRNA, the sequence of which is shown in SEQ ID No:4 or SEQ ID No:5, and the chemical formula of GSK2606414 is C 24 H 20 F3N5O, structural formula is

[0017] .

[0018] Furthermore, the leukemia mentioned is acute myeloid leukemia and acute lymphoblastic leukemia.

[0019] The beneficial effects of this invention are:

[0020] This invention confirms that BAHD1 is highly expressed in acute leukemia cells, thus using it as a molecular marker to accurately distinguish acute leukemia from healthy individuals by detecting the mRNA expression level of the BAHD1 gene, which is particularly suitable for early screening of infants and children. Furthermore, high BAHD1 expression is closely related to disease progression and prognosis, serving as an important indicator for monitoring treatment efficacy and predicting relapse, providing a scientific basis for developing personalized treatment plans. The development of diagnostic kits or detection methods based on BAHD1 will greatly improve the efficiency and accuracy of acute leukemia diagnosis.

[0021] This invention experimentally demonstrates that BAHD1 knockout significantly inhibits the proliferation and self-renewal of acute leukemia cells, while promoting their apoptosis and differentiation. In a mouse leukemia model, BAHD1 knockout effectively inhibited the proliferation of leukemia cells, reduced tumor burden and extramedullary invasion, and significantly prolonged survival. Furthermore, BAHD1 knockout also significantly enhanced the sensitivity of acute leukemia cells to traditional chemotherapy drugs and UPR inhibitors.

[0022] This invention creatively reveals that BAHD1 may influence the development, progression, relapse, and drug resistance of leukemia by regulating the classical UPR pathway. This confirms that targeting and inhibiting BAHD1 and its downstream pathways is a potentially effective new strategy for treating the development, relapse, and drug resistance of acute leukemia. Based on this, it is hoped that drugs targeting BAHD1 can be developed to enhance their inhibitory effect on the proliferation of acute leukemia cells. Combined use with existing chemotherapy drugs or other targeted therapies is expected to achieve synergistic effects and reduce drug resistance, improving treatment efficacy and safety. Attached Figure Description

[0023] Figure 1This is a comparison of protein expression levels of core epigenetic regulatory factors, including BAHD1, in hematologic malignancies and solid tumors, as shown in Example 1; A represents hematologic malignancies, and B represents solid tumors.

[0024] Figure 2 This is a comparison of BAHD1 expression levels between healthy individuals and individuals with acute leukemia in Example 1. A represents the GSE15061 database, and B represents the GSE2466 dataset.

[0025] Figure 3 Figure 2 shows the results of BAHD1 expression level and cell viability detection in patients with acute leukemia in Example 2. A represents BAHD1 expression level, and B represents cell viability comparison.

[0026] Figure 4 The image shows the Western blot results of BAHD1 protein expression in different types of acute leukemia cell lines in Example 3. A is KOPN-8, B is MOLM-13, C is SHI-1, and D is MV4.

[0027] Figure 5 This is a comparison of cell proliferation count results for different types of acute leukemia cell lines in Example 3. A is KOPN-8, B is MOLM-13, C is SHI-1, and D is MV4;11;

[0028] Figure 6 The images show the clone formation and number comparison of different types of acute leukemia cell lines in Example 3. A is KOPN-8, B is MOLM-13, C is SHI-1, and D is MV4;11;

[0029] Figure 7 This is a comparison of the proportion of apoptotic cells in different types of acute leukemia cell lines in Example 3. A is KOPN-8, B is MOLM-13, C is SHI-1, and D is MV4;11;

[0030] Figure 8 This is a comparison of the proportion of CD11b positive cells in different types of acute leukemia cell lines in Example 3. A is MOLM-13, B is SHI-1, and C is MV4;11.

[0031] Figure 9 The image shows the Western blot results of BAHD1 protein expression in KOPN-8 cells of each group in Example 4.

[0032] Figure 10 The images show a comparison of cell proliferation counts, colony formation, and apoptotic cell ratios in each group of KOPN-8 cells in Example 4; A represents cell proliferation, B represents photos and number comparisons of colony formation, and C represents the proportion of apoptotic cells.

[0033] Figure 11 The image shows the Western blot results of BAHD1 protein expression in KOPN-8 cells of each group in Example 5.

[0034] Figure 12 Comparative images of cell proliferation counts and colony formation in each group of KOPN-8 cells in Example 5; A shows cell proliferation, and B shows photos and comparisons of colony formation and their numbers;

[0035] Figure 13 The images show fluorescence images, survival time, and spleen volume comparisons of the control group and the BA_sg2 group of MOLM-13-derived mouse models in Example 6; A is an abdominal fluorescence image, B is a dorsal fluorescence image, C is a survival time comparison image, and D is a spleen volume comparison image.

[0036] Figure 14 The images show fluorescence images and survival comparisons of the control group and the BA_sg2 group MV4;11-derived mouse models in Example 6. A is an abdominal fluorescence image, B is a back fluorescence image, and C is a survival comparison.

[0037] Figure 15 This is a clustering heatmap obtained from RNA-seq sequencing of the BAHD1 knockout acute leukemia cell line KOPN-8 in Example 7.

[0038] Figure 16 The Venn diagram obtained from RNA-seq sequencing of KOPN-8 cells, a BAHD1 knockout acute leukemia cell line in Example 7, shows that A represents upregulated genes and B represents downregulated genes.

[0039] Figure 17 The scatter plot shows the enrichment of downregulated gene pathways in the BAHD1 knockout acute leukemia cell line KOPN-8 in Example 7. A represents apoptosis, B represents the regulation of intracellular signal transduction, C represents the regulation of cell death, D represents the regulation of protein modification, E represents the endoplasmic reticulum stress response, F represents the response to topologically incorrect proteins, G represents the cell response to topologically incorrect proteins, H represents the endoplasmic reticulum unfolded protein response, and I represents the endoplasmic reticulum unfolded protein response.

[0040] Figure 18 Example 7 shows the gene expression profile of key nodes in the UPR core pathway based on RNA-seq data;

[0041] Figure 19 This is a comparison of the mRNA levels of key molecules in the UPR pathway in BAHD1 knockout KOPN-8 cells in Example 7.

[0042] Figure 20This is a comparison of the levels of key molecules in the UPR pathway and the proportion of unfolded protein accumulation in BAHD1 knockout KOPN-8 cells in Example 7. A represents the protein level, and B represents the proportion of unfolded protein accumulation.

[0043] Figure 21 This is a comparison of the mRNA levels of key molecules in the UPR pathway in BAHD1 knockout MOLM13 cells in Example 7.

[0044] Figure 22 This is a comparison of the levels of key molecules in the UPR pathway and the proportion of unfolded protein accumulation in BAHD1 knockout MOLM13 cells in Example 7. A represents the protein level, and B represents the proportion of unfolded protein accumulation.

[0045] Figure 23 This is a comparison of the mRNA levels of key molecules in the UPR pathway in BAHD1 knockout SHI-1 cells in Example 7.

[0046] Figure 24 This is a comparison of the levels of key molecules in the UPR pathway and the proportion of unfolded protein accumulation in BAHD1 knockout SHI-1 cells in Example 7. A represents the protein level, and B represents the proportion of unfolded protein accumulation.

[0047] Figure 25 This is a comparison of the mRNA levels of key molecules in the UPR pathway in BAHD1 knockout MV4;11 cells in Example 7.

[0048] Figure 26 This is a comparison of the levels of key molecules in the UPR pathway and the proportion of unfolded protein accumulation in BAHD1 knockout MV4;11 cells in Example 7. A represents the protein level, and B represents the proportion of unfolded protein accumulation.

[0049] Figure 27 This is a comparison of cell viability of different acute leukemia cell lines in Example 8 after Ara-c treatment. A is KOPN-8, B is MOLM-13, C is SHI-1, and D is MV4;11;

[0050] Figure 28 This is a comparison of cell viability of different acute leukemia cell lines in Example 8 after treatment with VTP-50469. A is KOPN-8, B is MOLM-13, C is SHI-1, and D is MV4;11.

[0051] Figure 29 This is a comparison of cell viability of each acute leukemia cell line in Example 8 after treatment with STF-083010. A is KOPN-8, B is MOLM-13, C is MV4;11, and D is SHI-1.

[0052] Figure 30The image shows a comparison of cell viability of each acute leukemia cell line in Example 8 after treatment with GSK2606414. A is KOPN-8, B is MOLM-13, C is MV4;11, and D is SHI-1.

[0053] Figure 31 This is a comparison of cell viability in Example 9 between the control group and KOPN-8 acute leukemia cells after Ara-c treatment following endogenous BAHD1 overexpression.

[0054] Figure 32 This is a comparison of the cell viability of primary acute leukemia cells and drug-resistant KOPN-8 cells (AraR) treated with Ara-c in Example 9;

[0055] Figure 33 This is a comparison of the expression of BAHD1 and key proteins in the downstream UPR pathway in primary acute leukemia cells and drug-resistant KOPN-8 cells (AraR) in Example 9. A represents the BAHD1 protein level, and B represents the mRNA expression level of key proteins in the downstream UPR pathway.

[0056] Figure 34 This is a comparison of the cell viability of primary acute leukemia cells and drug-resistant MOLM-13 cells (AraR) treated with Ara-c in Example 9;

[0057] Figure 35 This is a comparison of the expression of BAHD1 and key proteins in the downstream UPR pathway in primary acute leukemia cells and drug-resistant MOLM-13 cells (AraR) in Example 9. A represents the BAHD1 protein level, and B represents the mRNA expression level of key proteins in the downstream UPR pathway.

[0058] Figure 36 This is a comparison of the levels of key proteins in the downstream UPR pathway in primary and drug-resistant acute leukemia cells (AraR) in Example 9. A represents KOPN-8, and B represents MOLM-13. Detailed Implementation

[0059] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0060] Example 1

[0061] This embodiment used the TCGA database to analyze the protein expression levels of core epigenetic regulatory factors, including BAHD1, in hematologic malignancies and solid tumors. Specifically, the core factors included BAHD1, BAHCC1, EED, CBX2, CBX4, CBX6, CBX7, and CBX8. The results are as follows: Figure 1 As shown, among classic H3K27me3 reader proteins, BAHD1, which contains only the BAH domain, exhibits relatively stable expression in solid tumors. However, in hematologic malignancies, its expression level increases with decreasing treatment response and disease relapse. This example compares the BAHD1 expression levels in healthy individuals and individuals with acute leukemia from the gene expression comprehensive databases GSE15061 and GSE2466. The results are as follows: Figure 2 As shown, BAHD1 is highly expressed in acute leukemia.

[0062] The data comparison above shows that BAHD1 is highly expressed in acute leukemia cells, thus it can serve as a molecular marker. By detecting the mRNA expression level of the BAHD1 gene, acute leukemia can be accurately distinguished from other types of leukemia. Furthermore, high BAHD1 expression is closely related to disease progression and prognosis, and can serve as an important indicator for monitoring treatment efficacy and predicting relapse, providing a scientific basis for the development of personalized treatment plans. The development of diagnostic kits or detection methods based on BAHD1 will greatly improve the efficiency and accuracy of acute leukemia diagnosis.

[0063] Furthermore, this embodiment provides a leukemia diagnostic kit containing reagents for detecting the mRNA expression level of the BAHD1 gene. The reagents for detecting the mRNA expression level of the BAHD1 gene include detection primers for the BAHD1 gene: the sequence of the upstream primer is shown in SEQ ID No:1, 5'-GCGACATCAGGACCAGAACA-3'; the sequence of the downstream primer is shown in SEQ ID No:2, 5'-GGTGGGGTGGAATAGTCTGC-3'.

[0064] Example 2

[0065] This embodiment uses siRNA technology to knock down the expression level of BAHD1 in patients with acute leukemia. The specific method is as follows:

[0066] I. Design of siRNA sequences targeting BAHD1 exons:

[0067] The si_BA shown in SEQ ID No:3 is 5'-GACAGAGAUAAGAAGUACUUCUUTA-3'; to comply with the WIPOST.26 standard, as shown in SEQ ID NO:3, T is used to replace U at positions 9, 16, 19, 20, 22, and 23 of the 5' end in RNA to represent uracil.

[0068] II. Extraction of mononuclear cells from peripheral blood of patients with acute leukemia:

[0069] First, aseptically collect peripheral venous blood from the patient using blood collection tubes containing an anticoagulant (such as EDTA or heparin). Gently invert the blood sample to mix it, preventing coagulation, but avoid vigorous shaking to prevent cell damage. Add an appropriate amount of lymphocyte separation medium with a density of 1.077 g / mL to the centrifuge tube. Then, carefully and slowly add anticoagulated whole blood to the surface of the separation medium, maintaining a clear interface between the two. Place the centrifuge tube in a centrifuge and centrifuge at 400 × g for 20-30 minutes at room temperature. After centrifugation, the liquid in the tube will separate into layers. The top layer is plasma and platelets, the middle layer is a transparent buffy coat, and the bottom layer is red blood cells and granulocytes. This buffy coat is rich in the peripheral blood mononuclear cells (PBMCs) we need. Carefully aspirate these cells with a pipette and transfer them to a new centrifuge tube. Add sufficient erythrocyte lysis buffer to the collected cell suspension, stop the reaction, centrifuge, add sufficient PBS buffer, mix thoroughly, centrifuge again, and discard the supernatant. Repeat this step 2-3 times to thoroughly remove the separation fluid, platelets, and residual plasma proteins.

[0070] III. siRNA infection of mononuclear cells from patients with acute leukemia:

[0071] Prepare two sterile centrifuge tubes: one tube contains a suitable amount of chemically synthesized siRNA diluted in serum-free medium (usual working concentration is 20 nM); the other tube contains an equal volume of serum-free medium diluted with the transfection reagent. After standing for five minutes, slowly add the diluted transfection reagent dropwise to the siRNA-containing solution, gently pipetting or vortexing to mix, and let stand at room temperature for 15-20 minutes to form the siRNA / transfection reagent complex. After the complex forms, evenly add it dropwise to a culture dish containing acute leukemia patient cells, gently shaking the dish to ensure even distribution. Incubate the cells at 37°C, 5% CO2 for 4-6 hours, then centrifuge and replace with normal complete culture medium to reduce cytotoxicity. Continue culturing the cells for 48 hours, then collect the cells and detect the BAHD1 silencing efficiency using RT-qPCR.

[0072] This embodiment further investigated the cell viability of BAHD1-silenced acute leukemia patients. The cell viability assay is as follows:

[0073] Cells to be tested were seeded into 96-well plates (100 μL per well, 30,000 cells / well), with NC group and BAHD1 silencing group (si_BA) set up. The plates were incubated at 37°C and 5% CO2 for 72 hours. Before detection, CellTiter-Glo reagent was equilibrated at room temperature for 30 minutes, and 20 μL of reagent was added to each well. The plates were shaken and mixed for 2 minutes to induce cell lysis, taking care to avoid interference from air bubbles between wells. The plates were then incubated at room temperature in the dark for 10 minutes to allow the luminescence signal to stabilize. The chemiluminescence value was then detected using a microplate reader, and the relative cell viability percentage of each group was calculated.

[0074] The results are as follows Figure 3 As shown, the cell viability of BAHD1-silenced acute leukemia patients was significantly lower than that of the control group, indicating that BAHD1 silencing effectively inhibits the viability of acute leukemia cells.

[0075] Example 3

[0076] In this embodiment, CRISPR / Cas9 technology was used to construct different BAHD1 knockout acute leukemia cell lines using artificially designed small nucleic acid BAHD1 gene silencing agent sgRNA.

[0077] In this embodiment, two small nucleic acid BAHD1 gene silencing agents sgRNA sequences targeting BAHD1 exons were designed using the CHOPCHOP online website: BA_sg1: 5'-GTACCCAGCCATTAGTGCGG-3' as shown in SEQ ID No:4; and BA_sg2: 5'-GTCGCCGCCGCACTAATGGC-3' as shown in SEQ ID No:5.

[0078] The specific method for constructing a BAHD1 knockout acute leukemia cell line using BA_sg1 (shown in SEQ ID No:4) as a gene silencing agent is as follows:

[0079] I. Synthesize two complementary oligonucleotide chains, Oligo 1 and Oligo 2, containing BsmbI sticky ends, according to the nucleotide sequence shown in SEQ ID No:4. Anneal the two oligonucleotide chains to form a double-stranded sgRNA fragment. The annealing system consisted of: 1 μL Oligo 1 (100 μM), 1 μL Oligo 2 (100 μM), 1 μL 10X T4 Ligation Buffer (NEB), 6.5 μL ddH2O, and 0.5 μL T4 PNK (NEB M0201S), for a total of 10 μL. Annealing conditions: 37°C for 30 minutes, 95°C for 5 minutes, then decreasing to 25°C at a rate of 5°C / minute. Finally, dilute the sgRNA fragment 1:200 for later use.

[0080] 2. Digestion of lentiCRISPR v2 plasmid with BsmbI: Take 1 μg of lentiCRISPR v2 plasmid, add 3 μL LFastDigest BsmBI (Fermentas), 3 μL FastAP (Fermentas), 6 μL 10X FastDigest Buffer (Fermentas), and 0.6 μL 100mM DTT (freshly prepared), and adjust the volume to 60 μL. Digest at 37℃ for 30 minutes. The digestion product was analyzed by DNA gel extraction, and the lentiCRISPRv2 plasmid was purified using a QIAquick colloid extraction kit.

[0081] III. Ligation reaction: Mix 50 ng of the purified plasmid from step II with 1 μL of the diluted sgRNA from step I, add 5 μL of 2X Quick Ligase Buffer (NEB), and bring the volume to 11 μL with dd water. Incubate overnight at 16°C.

[0082] IV. Transformation: Add 5 μL of the ligation product obtained in step III to 50 μL of competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, immediately place on ice for 5 minutes, add 250 μL of SOC medium, and incubate at 37°C with shaking for 1 hour. Spread on LB agar plates containing ampicillin and incubate overnight at 37°C. Pick single colonies, verify positive clones by PCR, and extract plasmids from positive clones using a plasmid extraction kit. Sequencing verification: Confirm correct sgRNA insertion using U6 promoter primers.

[0083] V. Lentiviral transfection of 293T cells: 293T cells were digested with trypsin at a concentration of 5 × 10⁻⁶ mcg / mL. 6Seed cells at a density of 70-80% per 10 cm dish (ensuring a cell density of 70-80% at transfection). Incubate overnight (16-24 hours) at 37°C and 5% CO2. Mix 20 μg of the target plasmid, 13.3 μg of psPAX2, and 6.7 μg of pMD2.G in 500 μL of Opti-MEM. Add 80 μL of PEI (1 mg / mL) to 500 μL of Opti-MEM, mix well, and incubate at room temperature for 5 minutes. Add the PEI solution dropwise to the plasmid mixture, vortex to mix, and incubate at room temperature for 15 minutes. Aspirate the old culture medium from the 293T cells and add 10 mL of fresh complete culture medium. Add the PEI-plasmid complex dropwise to the culture dish and gently shake to mix. Continue incubation at 37°C. 24 hours after transfection, aspirate the culture medium and replace it with 10 mL of fresh complete culture medium. 48 hours after transfection, collect the virus-containing supernatant into a 15 mL centrifuge tube and filter it through a 0.45 μm filter to remove cell debris. This collection can be repeated once after 24 hours, and the two virus solutions can be combined to increase yield.

[0084] VI. Lentiviral Infection of Cells: Acute leukemia cell lines KOPN-8 (MLL-ENL), MOLM-13 (MLL-AF9), SHI-1 (MLL-AF6), and MV4;11 (MLL-AF4) were constructed. The original culture medium for each cell line was removed, and infection medium containing virus and polybrene was added. The cells were gently mixed and incubated at 37°C. After 6-12 hours of infection, the medium was replaced with fresh complete medium. Puromycin selection was initiated 48-72 hours after infection and continued for 3-7 days until the uninfected control cells died completely. The resulting BAHD1 knockout acute leukemia cell line was designated as the BA_sg1 group.

[0085] Using the same method, a BAHD1 knockout acute leukemia cell line was constructed using BA_sg2 (shown in SEQ ID No:5) as the gene silencing agent, and labeled as the BA_sg2 group. The EV control group consisted of acute leukemia cell lines transfected with the empty vector lentiCRISPR v2 plasmid containing no target sgRNA sequence.

[0086] VII. After the stable cell lines were successfully constructed, proteins were extracted from the BA_sg1, BA_sg2 and EV groups. The expression of BAHD1 protein was examined by Western blot experiments to verify the knockout efficiency.

[0087] The specific method for Western blot experiments is as follows:

[0088] (1) Protein extraction and quantification: During the protein extraction process, the cell samples were washed with PBS and then directly added to RIPA lysis buffer for lysis. Protein extraction required incubation on ice for 30 minutes, with vortexing once every 10 minutes. Then, the samples were centrifuged at 12,000 rpm for 15 minutes at 4°C. Protein quantification was performed using the BCA method. A standard curve was prepared to ensure that the determination was within the linear range. The samples were adjusted to a uniform concentration according to the concentration.

[0089] (2) Gel preparation requires separate preparation of separating and stacking gels, mixed 1:1 to prepare suitable concentrations (mostly 7.5%). For sample preparation, protein samples should be mixed with 5× loading buffer at a 4:1 ratio, boiled at 100℃ for 5 minutes to denature the proteins, cooled, briefly centrifuged, and vortexed before loading. Electrophoresis conditions are: loading 20 μg total protein / lane, stacking gel electrophoresis at 80V constant voltage for approximately 30 minutes, and separating gel electrophoresis at 120V constant voltage for 60-90 minutes, until the bromophenol blue front is close to the bottom of the gel.

[0090] (3) Transfer: PVDF membrane pretreatment (activated with methanol for 1 minute, then rinsed with deionized water), filter paper and sponge pre-equilibrated in transfer buffer, and assembled in the order of black plate-sponge-filter paper-gel-membrane-filter paper-sponge-red plate. Prepare transfer buffer (1L): 3.03g Tris, 14.4g glycine, 200mL methanol, and deionized water to a final volume of 1000mL, adjusting the pH to 8.3. Transfer conditions: wet transfer system at 300mA constant current, ice bath transfer for 120 minutes.

[0091] (4) Membrane blocking: Block with 5% skim milk powder at room temperature for 2 hours. Wash three times with TBST for 5 minutes each time. Dilute the primary antibody according to the recommended dilution ratio and incubate overnight at 4°C with gentle agitation during incubation. Wash the secondary antibody three times with TBST for 10 minutes each time before incubation. Dilute the HRP-conjugated secondary antibody and incubate at room temperature for 2 hours with continuous agitation. Use ECL luminescent reagent for exposure detection using a chemiluminescence imaging system.

[0092] The results are as follows Figure 4 As shown, compared with the control group EV, the expression of BAHD1 protein in the BAHD1 knockout acute leukemia cell lines of the BA_sg1 group and BA_sg2 group was almost gone.

[0093] This embodiment further investigated the cell proliferation of cells in the BA_sg1 group, BA_sg2 group, and EV group. The cell proliferation experiment is as follows:

[0094] Cells were arranged at an appropriate density (e.g., 5 × 10⁶). 4Cells were seeded in 12-well plates, with 3 replicates per group, and incubated at 37°C in a 5% CO2 incubator. Starting from the day of seeding (Day 0), a sample was taken every 2 days for cell counting: after discarding the old culture medium, the cells were gently washed 1-2 times with PBS, and after thorough mixing, a small amount of cell suspension was taken for trypan blue staining and counting. The number of viable cells was recorded and the cell concentration (cells / mL) was calculated. At the same time, a growth curve was plotted to observe the proliferation trend.

[0095] The results are as follows Figure 5 As shown, compared with the control group EV, the cell number growth of BAHD1 knockout acute leukemia cell lines in the BA_sg1 group and BA_sg2 group was significantly slower, indicating that BAHD1 knockout effectively inhibits the proliferation of acute leukemia cells.

[0096] This embodiment further investigated the colony formation of cells in the BA_sg1 group, BA_sg2 group, and EV group. The colony formation experiment is as follows:

[0097] Prepare a bottom layer medium containing 0.5% low-melting-point agarose (the complete medium and agarose are mixed in proportion, cooled to 40°C in a water bath, and then added to a 6-well plate at 1.5 mL per well, allowing it to solidify at room temperature). Simultaneously, prepare a top layer medium containing 0.3% agarose and maintain it in a 37°C water bath. After counting the suspension cells in the logarithmic growth phase, mix them with the top layer medium at a density of 5000 cells / well and quickly spread them onto the solidified bottom layer agar (2 mL per well). After solidification at room temperature, transfer the plate to a 37°C, 5% CO2 incubator for 2-3 weeks. During this period, observe the colony formation regularly and count and stain cell clusters with a diameter >50 μm using an inverted microscope. Finally, calculate the number of colonies formed.

[0098] The results are as follows Figure 6 As shown, compared with the control group EV, the number of clones formed by BAHD1 knockout acute leukemia cell lines in the BA_sg1 group and BA_sg2 group was significantly reduced, indicating that BAHD1 knockout significantly inhibited the cell self-renewal capacity.

[0099] This embodiment further investigated the apoptosis of cells in the BA_sg1 group, BA_sg2 group, and EV group. The flow cytometry assay for apoptosis is as follows:

[0100] Apoptosis was detected using Annexin V-FITC / PI double staining. Cells in the logarithmic growth phase were first collected, washed twice with PBS, and then the cell density was adjusted to 1×10⁶. 6Cells / mL, take 100 μL of cell suspension and add it to a flow cytometry tube; set up a blank control (unstained cells), a single-stained control (labeled with Annexin V or PI respectively), and an experimental group. Add 5 μL of Annexin V-FITC and 5 μL of PI staining solution respectively, incubate at room temperature in the dark for 15 minutes, and then immediately add 400 μL of Binding Buffer and mix well. Analyze the cells within 1 hour.

[0101] The results are as follows Figure 7 As shown, compared with the control group EV, the proportion of apoptotic cells in BAHD1 knockout acute leukemia cell lines in the BA_sg1 group and BA_sg2 group was significantly increased, indicating that BAHD1 knockout can promote apoptosis of acute leukemia cells.

[0102] This embodiment further investigated the cell differentiation of cells in the BA_sg1 group, BA_sg2 group, and EV group. The cell differentiation experiments are as follows:

[0103] Cell differentiation was detected using CD11b staining. Cells in the logarithmic growth phase were first collected, washed twice with PBS, and then the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL: Add 100 μL of cell suspension to a flow cytometry tube; set up a blank control (unstained cells) and an experimental group; add 5 μL of CD11b staining solution, incubate at room temperature in the dark for 15 minutes, then centrifuge at 300g for 5 minutes. Add 400 μL of PBS and mix well; analyze within 1 hour.

[0104] The results are as follows Figure 8 As shown, compared with the control group EV, the expression level of CD11b, a myeloid differentiation marker, was significantly enhanced in the BA_sg1 group and the BA_sg2 group of BAHD1 knockout acute leukemia cell lines, indicating that BAHD1 knockout promotes the differentiation ability of acute leukemia cells.

[0105] Example 4

[0106] In this embodiment, the BAHD1 knockdown acute leukemia cell line KOPN-8 (MLL-ENL) was constructed using the shRNA system. Using the pLKO.1 vector, the shRNA sequence targeting the BAHD1 gene was cloned via EcoRI and AgeI restriction endonuclease sites to construct the shRNA expression plasmid. The specific steps were the same as those used in the CRISPR / Cas9 gene editing method in Example 3. The EV control group consisted of cells transfected with the empty vector lentiCRISPR v2 plasmid containing the target sgRNA sequence.

[0107] The shRNA sequence in this embodiment is:

[0108] SEQ ID No:6 shows BA_sh1: 5'-CCCAGAAAGTTAACTGATGAT-3'; or SEQ ID No:7 shows BA_sh2: 5'-CAAACCTCCCAGCGGTTCTAA-3'.

[0109] This embodiment further investigated the expression of BAHD1 protein, cell proliferation, colony formation, and apoptosis in the BAHD1 knockdown acute leukemia cell line KOPN-8. The results are as follows: Figures 9-10 As shown, compared with the control group EV, BAHD1 knockdown reduced BAHD1 protein expression, significantly inhibited cell proliferation and self-renewal capacity, and promoted cell apoptosis.

[0110] Example 5

[0111] In this embodiment, the KOPN-8 (MLL-ENL) cell line of acute leukemia with endogenous overexpression of BAHD1 was constructed using CRISPR / Cas9 technology.

[0112] I. Designing sgRNA sequences targeting BAHD1 using the CHOPCHOP online website:

[0113] BA_asg1 shown in SEQ ID No: 8: 5'-CACCTGGGTCGGTCGTCCCAACGC-3'; or BA_asg2 shown in SEQ ID No: 9: 5'-CACCGACGCAGCAGCGTGGAGCCC-3'.

[0114] Two complementary oligonucleotide chains containing BsmbI sticky ends were synthesized. The sgRNA was then annealed.

[0115] 2. The lentiCRISPR SAM plasmid was digested with BsmbI and then purified.

[0116] 3. Perform a ligation reaction, transform the reaction product, and sequence it to verify whether the construction was successful.

[0117] IV. CRISPRa infection of leukemia cells involves two steps: 293T cells are transfected with MPH plasmid lentivirus, followed by viral infection of leukemia cells. After drug screening to obtain stable cell lines, SAM plasmid lentivirus infection is performed again for secondary drug screening. The EV control group consists of cells transfected with the empty vector lentiCRISPR SAM plasmid containing the target sgRNA sequence.

[0118] V. After the stable cell line was successfully constructed, Western blot experiments were used to verify the overexpression efficiency.

[0119] The results are as follows Figures 11-12The results showed that, compared with the control group EV, BAHD1 overexpression significantly enhanced the proliferation rate of acute leukemia cells, and the number and size of clones were significantly increased, indicating that BAHD1 overexpression enhanced the cell's proliferative capacity and self-renewal capacity.

[0120] Example 6

[0121] This embodiment constructs a luciferase-labeled BAHD1 knockout acute leukemia cell-derived xenograft tumor mouse model: the luciferase CDX model. The specific construction method is as follows:

[0122] Following the method described in Example 3, stable transgenic strains MOLM-13 (MLL-AF9) and MV4;11 (MLL-AF4) expressing luciferase were constructed via lentiviral transfection using the BA_sg1 sequence shown in SEQ ID No:3 and the BA_sg2 sequence shown in SEQ ID No:4. After screening with antibiotic (G418), the luminescence intensity was verified by in vitro bioluminescence imaging (BLI). Subsequently, 1×10 6 -3×10 6 Luciferase-positive cells in the logarithmic growth phase were injected via tail vein into 6-8 week old immunodeficient mice (Cyagen (Suzhou) Biotechnology Co., Ltd.). The EV control group consisted of mice with BAHD1 non-knockout MOLM-13 (MLL-AF9) and MV4;11 (MLL-AF4) models. Tumor growth was monitored weekly using a small animal in vivo imaging system (IVIS) 10 minutes after intraperitoneal injection of 150 mg / kg D-fluorescein (dissolved in PBS).

[0123] The results are as follows Figures 13-14 As shown, the fluorescence intensity of BAHD1 knockout mice was significantly reduced; simultaneously, anatomical observation revealed that the spleen volume of the BAHD1 knockout group was significantly smaller than that of the control group, and the survival time was significantly prolonged. These results indicate that BAHD1 knockout inhibits the proliferation of leukemia cells in mice, reduces extramedullary infiltration and overall tumor burden, thereby prolonging survival.

[0124] Example 7

[0125] In this embodiment, RNA-seq of the BAHD1 knockout acute leukemia cell line KOPN-8 (MLL-ENL) constructed in Example 3 was performed, and enrichment analysis was conducted based on the gene ontology (GO) of the downregulated genes in the sequencing results, aiming to reveal the potential molecular mechanism by which BAHD1 regulates the occurrence and development of leukemia.

[0126] The results are as follows Figures 15-17As shown, after BAHD1 knockout, the proportions of upregulated and downregulated genes among differentially expressed genes were similar, which differs from the known H3K27me3 reader function of BAHD1, suggesting that it may have an important but under-recognized function in acute leukemia. Pathway enrichment analysis of the downregulated genes showed that these genes were significantly enriched in endoplasmic reticulum stress-related pathways such as the UPR pathway.

[0127] Based on the gene expression profiles of key nodes in the UPR core pathway using RNA-seq data, this example further examined the expression changes of key molecules in the UPR pathway at the mRNA and protein levels in different acute leukemia cell models constructed in Example 3: KOPN-8 (MLL-ENL), MOLM13 (MLL-AF9), SHI-1 (MLL-AF6), and MV4;11 (MLL-AF4), as well as the detection of unfolded proteins.

[0128] The level of unfolded proteins in cells was detected using the TPE-MI staining method (TMI method).

[0129] First, cells in the logarithmic growth phase were collected, washed twice with PBS, and then the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL, take 100μL of cell suspension and add it to a flow cytometer; set up blank control (unstained cells) and experimental group, add TPE-MI staining solution to a final concentration of 2mM, incubate at 37℃ for 45 minutes in the dark, centrifuge at 300g for 5 minutes, discard the supernatant, wash twice with PBS, add 400μL of PBS and mix well, and perform detection within 1 hour.

[0130] The results are as follows Figures 18-26 As shown, after BAHD1 knockdown, transcriptome analysis revealed a significant downregulation of expression in all UPR pathway-related genes. RT-qPCR and Western blotting further confirmed that the expression of key UPR pathway genes was significantly reduced at both the mRNA and protein levels. Unfolded protein assays showed a significant increase in its level, indicating that impaired UPR pathway function leads to the accumulation of unfolded proteins.

[0131] Example 8

[0132] This example investigated the sensitivity of BAHD1 knockout acute leukemia cell lines to conventional chemotherapy drugs (Ara-c) and menin-KMT2A interaction inhibitors (VTP-50469).

[0133] In this embodiment, BAHD1 knockout acute leukemia cell lines KOPN-8 (MLL-ENL), MOLM-13 (MLL-AF9), SHI-1 (MLL-AF6), and MV4;11 (MLL-AF4) were constructed using the BAHD1 gene silencing agent sgRNA. The specific construction method was the same as in Example 3. Cell lines knocked out by BA_sg1 (SEQ ID No:4) were labeled as the BA_sg1 group, and cell lines knocked out by BA_sg2 (SEQ ID No:5) were labeled as the BA_sg2 group. The EV control group consisted of cells transfected with the empty vector lentiCRISPR v2 plasmid containing the target sgRNA sequence. Ara-c used in this embodiment was purchased from MCE (catalog number HY-13605); VTP-50469 was purchased from MCE (catalog number HY-114162).

[0134] This embodiment uses CTG to detect cell viability:

[0135] BAHD1 knockout acute leukemia cell lines KOPN-8 (MLL-ENL), MOLM-13 (MLL-AF9), SHI-1 (MLL-AF6), and MV4;11 (MLL-AF4) from the BA_sg1 and BA_sg2 groups, along with EV control cells, were seeded in 96-well plates (100 μL per well, 5000 cells / well). Different drug concentrations were used in the experimental groups to assess the IC50 of each acute leukemia cell line. 50 .

[0136] (1) The concentration (nM) of the chemotherapeutic drug Ara-c in each group of test cells is as follows:

[0137] KOPN-8 (MLL-ENL) cells: 0, 100, 250, 500, 1000, 2000;

[0138] MOLM-13 (MLL-AF9) cells: 0, 100, 250, 500, 1000, 2000, 4000;

[0139] SHI-1 (MLL-AF6) cells: 0, 100, 250, 500, 1000, 2000, 4000;

[0140] MV4;11(MLL-AF4) cells: 0, 100, 250, 500, 1000, 2000, 4000.

[0141] (2) The concentration (nM) of the menin-KMT2A interaction inhibitor VTP-50469 in each group of test cells is as follows:

[0142] KOPN-8 (MLL-ENL) cells: 0, 50, 100, 200, 500, 1000;

[0143] MOLM-13 (MLL-AF9) cells: 0, 50, 100, 250, 500, 1000;

[0144] SHI-1 (MLL-AF6) cells: 0, 250, 500, 1000, 2000, 4000;

[0145] MV4;11(MLL-AF4) cells: 0, 2.5, 5, 10, 20.

[0146] The cells were incubated at 37°C and 5% CO2 for 48 or 72 hours, respectively. Before detection, CellTiter-Glo reagent was equilibrated at room temperature for 30 minutes, and 20 μL of reagent was added to each well. The mixture was shaken for 2 minutes to induce cell lysis, taking care to avoid air bubbles between wells. The cells were then incubated at room temperature in the dark for 10 minutes to stabilize the chemiluminescence signal. Subsequently, the chemiluminescence value was detected using a microplate reader, and the relative cell viability percentage of each experimental group was calculated. The IC50 of acute leukemia cells in each group was measured. 50 like Figure 27 , Figure 28 As shown in Tables 1 and 2.

[0147] Table 1

[0148]

[0149] Table 2

[0150]

[0151] The results showed that BAHD1 knockout significantly reduced the IC50 of acute leukemia cells. 50 This indicates that BAHD1 knockout enhances the sensitivity of cells to conventional chemotherapy drugs and menin-KMT2A interaction inhibitors.

[0152] This embodiment further investigated the sensitivity of BAHD1 knockout acute leukemia cell lines KOPN-8 (MLL-ENL) cells, MOLM-13 (MLL-AF9) cells, SHI-1 (MLL-AF6) cells, and MV4;11 (MLL-AF4) cells, as well as EV control group cells, to IRE1 inhibitor (STF-083010) and PERK inhibitor (GSK2606414).

[0153] The STF-083010 used in this embodiment was purchased from MCE, with part number HY-15845; GSK2606414 was purchased from TargetMol, with part number T2614.

[0154] This embodiment uses CTG to detect cell viability:

[0155] BAHD1 knockout acute leukemia cell lines KOPN-8 (MLL-ENL), MOLM-13 (MLL-AF9), SHI-1 (MLL-AF6), and MV4;11 (MLL-AF4) from the BA_sg1 and BA_sg2 groups, along with EV control cells, were seeded in 96-well plates (100 μL per well, 5000 cells / well). Different drug concentrations were used in the experimental groups to assess the IC50 of each acute leukemia cell line. 50 .

[0156] (1) The IRE1 inhibitor (STF-083010) concentration (μM) of each group of test cells is as follows:

[0157] KOPN-8 (MLL-ENL) cells: 0, 6.25, 12.5, 25, 50, 100;

[0158] MOLM-13 (MLL-AF9) cells: 0, 6.25, 12.5, 25, 50, 100;

[0159] SHI-1 (MLL-AF6) cells: 0, 6.25, 12.5, 25, 50, 100;

[0160] MV4;11(MLL-AF4) cells: 0, 6.25, 12.5, 25, 50, 100.

[0161] (2) The PERK inhibitor (GSK2606414) concentration (μM) of each group of test cells is as follows:

[0162] KOPN-8 (MLL-ENL) cells: 0, 1.25, 2.5, 5, 10, 20;

[0163] MOLM-13 (MLL-AF9) cells: 0, 1.25, 2.5, 5, 10, 20;

[0164] SHI-1 (MLL-AF6) cells: 0, 1.25, 2.5, 5, 10, 20;

[0165] MV4;11(MLL-AF4) cells: 0, 1.25, 2.5, 5, 10, 20.

[0166] IC50 of various acute leukemia cells were measured 50 like Figure 29 , Figure 30 As shown in Tables 3 and 4.

[0167] Table 3

[0168]

[0169] Table 4

[0170]

[0171] The results showed that after BAHD1 knockout, acute leukemia cells IC50 decreased. 50 Significantly reduced, BAHD1 knockout can enhance the killing effect of IRE1 / PERK inhibitors on acute leukemia cells.

[0172] Example 9

[0173] This example investigated the sensitivity of the control group constructed in Example 5 and the KOPN-8 (MLL-ENL) acute leukemia cell line with endogenous BAHD1 overexpression to conventional chemotherapy drug (Ara-c) at concentrations of 1, 250, 500, 1000, 2000, and 4000 nM. The results are as follows: Figure 31 As shown, the control group IC 50 The IC in group BA_asg1 is 497.5nM. 50 The IC group BA_asg2 has a capacity of 1467nM. 50 The concentration was 1612 nM. This indicates that endogenous overexpression of BAHD1 significantly increased the IC50 of acute leukemia cells. 50 This suggests that high BAHD1 expression may be associated with resistance to traditional chemotherapy drugs in acute leukemia.

[0174] Therefore, this embodiment further established an acute leukemia drug-resistant strain (AraR) through continuous induction with low concentrations of Ara-c. The specific construction method is as follows:

[0175] KOPN-8 (MLL-ENL) and MOLM-13 (MLL-AF9) leukemia cell lines in logarithmic growth phase were selected and seeded in 6-well plates (density 1×10⁻⁶) with standard medium (RPMI-1640 + 10% FBS). 5 Cells / mL were added, along with an initial low concentration of chemotherapy drug (Ara-c concentration of 10 nM). Cell status was observed every 48 hours, and fluid was replenished to maintain the drug concentration. Once cells regained stable proliferation, the drug concentration was increased in increments of 20%-50% of the initial concentration, maintaining each concentration until cells could proliferate normally at that concentration. This escalation process was continued for 6-12 months until the lethal concentration (IC50 / mL for normal cells) was finally obtained.50 The drug-resistant strain was stably grown at a concentration 5-10 times higher than that of the parent strain, and intermediate passages of cells needed to be periodically cryopreserved as backups. The IC50 values ​​between the parent and drug-resistant strains were determined using the CTG assay. 50 Differences, results as Figure 32 and Figure 34 As shown, IC50 of KOPN-8 primary cells 50 The IC50 of the drug-resistant cell model was 150 nM. 50 The IC50 value for MOLM-13 primary cells was 900 nM. 50 The IC50 of the drug-resistant cell model was 200 nM. 50 >10000nM; successful construction of the drug-resistant strain is verified.

[0176] This embodiment investigated the expression levels of BAHD1 and its downstream target pathways in Ara-c resistant cells. RT-qPCR and Western blotting results are as follows: Figure 33 , Figure 35 and Figure 36 As shown, the expression of BAHD1 and key proteins in its downstream UPR pathway were consistently and significantly upregulated in drug-resistant strains. These data suggest that BAHD1 may regulate drug resistance in acute leukemia by activating the UPR pathway.

Claims

1. The application of BAHD1 gene expression inhibitors in the preparation of leukemia treatment drugs, characterized in that, The leukemia is acute myeloid leukemia and acute lymphoblastic leukemia. The BAHD1 gene expression inhibitor is a man-made small nucleic acid BAHD1 gene silencing agent. The man-made small nucleic acid BAHD1 gene silencing agent is siRNA, sgRNA or shRNA. The sequence of the siRNA is shown in SEQ ID No:3; the sequence of the sgRNA is shown in SEQ ID No:4 or SEQ ID No:5; and the sequence of the shRNA is shown in SEQ ID No:6 or SEQ ID No:

7.

2. The application of the BAHD1 gene expression inhibitor according to claim 1 in the preparation of leukemia treatment drugs, characterized in that, The leukemia treatment drug is a combination of a man-made small nucleic acid BAHD1 gene silencing agent and cytarabine. The man-made small nucleic acid BAHD1 gene silencing agent is sgRNA, and the sequence of the sgRNA is shown in SEQ ID No:4 or SEQ ID No:

5.

3. The application of the BAHD1 gene expression inhibitor according to claim 1 in the preparation of leukemia treatment drugs, characterized in that, The leukemia treatment drug is a combination of a man-designed small nucleic acid BAHD1 gene silencing agent and a menin-KMT2A interaction inhibitor, VTP50469. The man-designed small nucleic acid BAHD1 gene silencing agent is an sgRNA, the sequence of which is shown in SEQ ID No:4 or SEQ ID No:

5. The chemical formula of VTP50469 is C0. 32 H 47 FN6O4S, structural formula is 。 4. The application of the BAHD1 gene expression inhibitor according to claim 1 in the preparation of leukemia treatment drugs, characterized in that, The leukemia treatment drug is a combination of a man-made small nucleic acid BAHD1 gene silencing agent and an IRE1 inhibitor STF-083010. The man-made small nucleic acid BAHD1 gene silencing agent is sgRNA, and the sequence of the sgRNA is shown in SEQ ID No:4 or SEQ ID No:

5. The chemical formula of STF-083010 is C 15 H 11 NO3S2, structural formula is 。 5. The application of the BAHD1 gene expression inhibitor according to claim 1 in the preparation of leukemia treatment drugs, characterized in that, The leukemia treatment drug is a combination of a man-made small nucleic acid BAHD1 gene silencing agent and a PERK inhibitor GSK2606414. The man-made small nucleic acid BAHD1 gene silencing agent is sgRNA, and the sequence of the sgRNA is shown in SEQ ID No:4 or SEQ ID No:

5. The chemical formula of GSK2606414 is C 24 H 20 F3N5O, structural formula is 。

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