Use of p300 / cbp inhibitors in the preparation of a medicament for treating igh::dux4 rearranged leukemia

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a p300 / CBP inhibitor in preparation of a drug for treating IGH::DUX4 rearranged leukemia. The research is based on IGH::DUX4 IGH::DUX4 IGH::DUX4 IGH:: the interaction relationship between the fusion protein and p300 / CBP, and the inhibition effect of the p300 / CBP inhibitor on the leukemia cell of the subtype is verified by combining cell experiments and animal models, so that the existing technical blank is filled, and a new targeted treatment option is provided for the refractory subtype.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a p300 / CBP inhibitor in the preparation of a treatment... IGH::DUX4 Application in drugs for rearranged leukemia. Background Technology

[0002] Acute B-lymphoblastic leukemia (B-ALL) is a malignant hematologic malignancy originating from B-lineage progenitor cells. It is a common hematologic malignancy in children, adolescents, and young adults. Different molecular subtypes show significant differences in pathogenesis, response to drug treatment, and long-term prognosis. IGH::DUX4 B-ALL with rearrangements (also broadly referred to as DUX4 / IGH rearrangements B-ALL) is a distinct subtype of B-ALL with characteristic molecular markers; the core genetic event of this subtype is chromosome 4. DUX4 Gene fragment inserted into the immunoglobulin heavy chain on chromosome 14 IGH locus ， Gene rearrangements can occur through two forms: chromosomal translocation and fragment insertion; these rearrangements produce aberrant chimeric transcripts, which are continuously and abnormally expressed. IGH::DUX4 Fusion protein, which is a core oncogenic factor driving the occurrence and progression of leukemia. IGH::DUX4 Fusion proteins, as aberrant transcription factors, can disrupt the overall transcriptional regulatory network of cells on a large scale. On the one hand, they induce abnormal activation of downstream target genes, and on the other hand, they cause RNA alternative splicing disorder. Typically, this is manifested by the large accumulation of ERGalt aberrant splice bodies, which modifies the malignant biological phenotype of leukemia cells at the molecular level.

[0003] Epidemiological data show that IGH::DUX4 Rearranged B-ALL accounts for 5%–10% of all B-ALL cases; the incidence rate in children is 4%–7%, with a higher detection rate in adolescents and young adults (AYA). Some clinical statistics show that this subtype accounts for up to 18.5% in this population, making it one of the most common fusion gene subtypes of B-ALL in this age group. This subtype has unique clinical prognostic characteristics: patients show poor early minimal residual disease (MRD) clearance after conventional chemotherapy and have a poor initial treatment response, but long-term survival is good after receiving standard intensive combination chemotherapy, with a high 5-year overall survival rate. Currently, first-line treatment for this subtype still mainly relies on traditional multi-drug combination chemotherapy. Long-term chemotherapy easily leads to multiple adverse reactions: short-term acute toxicities such as myelosuppression, severe infection, liver and kidney damage, and anthracycline-induced cardiotoxicity can occur; long-term complications such as reproductive dysfunction, endocrine disorders, and secondary tumors can occur. For children and adolescents who are in a critical stage of growth and development, reducing the intensity of chemotherapy exposure and developing highly selective targeted drugs are urgent clinical needs to be addressed in this field.

[0004] p300 and CBP (CREB-binding protein) are highly homologous histone acetyltransferases (HATs), belonging to the p300 / CBP family, and are often collectively referred to as p300 / CBP. They are key transcriptional coactivators in eukaryotes, widely involved in core physiological processes such as cell cycle, proliferation, differentiation, and apoptosis. The p300 / CBP complex can catalyze histone acetylation to remodel chromatin conformation and regulate the activity of various key transcription factors. Abnormal function of the p300 / CBP complex is closely related to the occurrence, invasion, and drug resistance of solid tumors and hematological malignancies, making it a highly valuable target for cancer targeted therapy. Current basic research confirms that wild-type DUX4 protein can specifically bind to and recruit the p300 / CBP complex through its C-terminal domain, mediating extensive hyperacetylation of histone H3, thereby initiating the transcriptional program of downstream oncogenes.

[0005] Currently, there are no specifically approved [treatments / treatments] globally. IGH::DUX4 Targeted therapy for rearranged B-ALL. Current functional studies of DUX family proteins largely focus on solid tumors. For example, in studies related to CIC-DUX4 fusion sarcoma and facioscapulohumeral muscular dystrophy (FSHD), it has been demonstrated that the p300 / CBP inhibitor iP300w can block the interaction between wild-type DUX4, CIC-DUX4 fusion proteins, and p300 / CBP, reverse abnormal histone acetylation modifications, and inhibit malignant proliferation of tumor cells. iP300w's CAS number is 1889284-33-6, and its molecular formula is C... 29 H 27 F5N6O4, the structural formula is as follows: , InChI Key: SWXCHCQCWFXILM-NPPGMGHMSA-N, SMILES string: O=C(NC1(C2=O)CC (C3=C1C=CC(C4=CN(N=C4)CC(NC)=O)=C3)F)N2CC(N(CC5=CC=C(C=C5)F)C(C(F)(F)F)C)=O.

[0006] against IGH::DUX4 Rearrangement of hematologic malignancy subtypes: Existing literature only provides a preliminary explanation. IGH::DUX4 The pathogenic mechanism of gene rearrangement (DUX4-r) has been confirmed. The truncated fusion protein produced by this gene rearrangement alters its binding ability to p300 / CBP, and its oncogenic effect is instead mediated by the GTF2I protein. However, current research has not yet fully elucidated this mechanism. IGH::DUX4 The regulatory relationship between the fusion protein and p300 / CBP.

[0007] In conclusion, IGH::DUX4Clinical treatment for rearranged acute B-lymphoblastic leukemia relies solely on intensive chemotherapy regimens with significant toxic side effects. The field lacks both specific targeted therapies for this subtype and comprehensive treatment options. IGH::DUX4 Work related to targeted drug development along the p300 / CBP axis of the fusion protein. Summary of the Invention

[0008] The purpose of this disclosure is to provide a p300 / CBP inhibitor in the preparation of a treatment... IGH::DUX4 The application of drugs in the treatment of rearrangement leukemia is based on this study. IGH::DUX4 The interaction between the fusion protein and p300 / CBP, combined with cell experiments and animal models to verify the inhibitory effect of p300 / CBP inhibitors on this subtype of leukemia cells, fills the gap in existing technology and provides a new targeted therapy option for this refractory subtype.

[0009] The objective of this disclosure is achieved through the following technical solution: In a first aspect of this disclosure, this disclosure provides a p300 / CBP inhibitor in the preparation of a treatment for... IGH:: DUX4 Application in drugs for rearranged leukemia.

[0010] In some specific embodiments of this disclosure, the p300 / CBP inhibitor is a HAT catalytic domain inhibitor; optionally, the HAT catalytic domain inhibitor is a selective small molecule inhibitor; optionally, the selective small molecule inhibitor is iP300w.

[0011] In some specific embodiments of this disclosure IGH::DUX4 The fusion protein lacks the wild-type DUX4 C-terminal activation domain and functionally interacts with the TAZ1 and / or TAZ2 domains of the CBP / p300 protein in a non-C-terminal dependent manner. The iP300w blocks... IGH::DUX4 The interaction between the fusion protein and p300 / CBP inhibits... IGH::DUX4 Aberrant transcriptional programs mediated by fusion proteins.

[0012] In some specific embodiments of this disclosure, the iP300w is capable of suppressing p300 / CBP-mediated [therapeutic effects]. IGH:: DUX4 The expression of downstream target genes of the fusion protein is inhibited. IGH::DUX4 Fusion protein-induced oncogenic RNA alternative splicing.

[0013] In some specific embodiments of this disclosure, the oncogenic RNA alternative splice includes an ERGalt aberrant splice variant, and the iP300w can significantly reduce the mRNA expression of ERGalt, and its inhibitory effect on ERGalt is better than that of vincristine.

[0014] In some specific embodiments of this disclosure, the drug acts on IGH::DUX4 In a PDX model derived from patients with rearranged acute B-lymphoblastic leukemia, it can reduce the proportion of human leukemia cells in the peripheral blood of model mice, alleviate leukemia-induced splenomegaly, and relieve diffuse infiltration of leukemia cells in the spleen and repair spleen tissue structure.

[0015] In some specific embodiments of this disclosure, the active ingredient of the drug is iP300 or a pharmaceutically acceptable salt thereof; optionally, the drug further includes a pharmaceutically acceptable carrier or excipient; optionally, the drug is an injectable formulation.

[0016] In some specific embodiments of this disclosure, the drug is used alone or in combination with other drugs.

[0017] In a second aspect, this disclosure provides a method for treating IGH::DUX4 A pharmaceutical composition for rearranged acute B-lymphoblastic leukemia, comprising a therapeutically effective amount of iP300w or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0018] In a third aspect, this disclosure provides an in vitro screening method. IGH::DUX4 The method for targeting inhibitors of rearranged acute B-lymphoblastic leukemia includes the following steps: (1) Constructing expressions IGH::DUX4 The precursor B lymphocyte model of the fusion protein was set up without... DUX4 Rearranged B-ALL cells served as a negative control; (2) After co-incubating the candidate compound with the cells in (1), the expression levels of downstream target genes of DUX4, the abundance of ERGalt splicing genes, and other parameters were detected. IGH::DUX4 The binding affinity of the fusion protein to CBP / p300; (3) Select compounds that meet at least one of the following effects as candidate drugs: reduce IGH::DUX4 The fusion protein exhibits strong binding affinity to CBP / p300, downregulates ERGalt splice abundance, and modulates the expression levels of downstream target genes of DUX4; the candidate drug can specifically inhibit... IGH::DUX4 Positive cell proliferation indicates the presence of the target compound.

[0019] In some embodiments of this disclosure, the protein interaction detection method in step (2) is selected from at least one of Co-IP, FRET, and PLA (Parallel Linkage Assay); the gene and splice assay is performed using at least one of RT-qPCR, RNA sequencing, and Western blot (WB).

[0020] The technical solution provided in this disclosure has the following technical contributions: (1) This disclosure establishes for the first time the relationship between the p300 / CBP inhibitor iP300w and... IGH::DUX4 Treatment links for rearranged acute B-lymphoblastic leukemia, opening up IGH::DUX4 The novel technical pathway for the intervention of this subtype of disease, based on the molecular action axis of the fusion protein-p300 / CBP, confirms that iP300w can serve as a targeted active substance for this specific leukemia subtype, providing a new candidate direction for the development of clinical targeted drugs. (2) This disclosure uses mammalian two-hybrid experiments to verify at the molecular level IGH::DUX4 The fusion protein exhibits a clear interaction with p300 / CBP, with the core binding region identified as the TAZ1 domain. The TAZ2 domain also demonstrated strong binding ability. These findings clarify the key target region for drug action, providing a direct molecular mechanism basis for the targeted screening and application of p300 / CBP inhibitors. (3) This disclosure is in IGH::DUX4 Knockdown of EP300 and CREBBP genes in positive NALM6 cells confirmed that p300 / CBP proteins are involved in regulation. IGH::DUX4 This involves downstream gene transcription and alternative splicing of oncogenic RNA mediated by p300. Experimental results showed that knocking out the aforementioned genes significantly downregulated the expression levels of characteristic aberrant splice variants such as ERGalt and related oncogenic target genes, demonstrating that p300 / CBP is not solely associated with oncogenic RNA. IGH::DUX4 It involves not only physical binding, but also deep involvement in the regulation of malignant phenotypes in leukemia, clarifying that this molecular axis has a targeted functional value. (4) Cell viability assay results confirmed that iP300w could effectively inhibit cell death in a time- and dose-dependent manner. IGH:: DUX4 Proliferation of positive NALM6 leukemia cells was observed. However, the drug did not exhibit the same killing effect against other B-ALL subtypes such as REH, SEM, and SUPB, demonstrating good subtype selectivity. The calculated half-maximal inhibitory concentration (IC50) for NALM6 cells after 48 hours of treatment was [value missing]. 50 The value was 1234 nM, with a 95% confidence interval of 886–1786 nM, which quantified the in vitro efficacy of the drug against target cells. (5) RT-qPCR results showed that iP300w could be significantly downregulated IGH::DUX4The mRNA expression of ERGalt, a characteristic abnormal splice variant of leukemia, was inhibited. It showed stable inhibitory effects at multiple time points, including 12 h, 24 h, and 48 h, and its regulatory effect on ERGalt was superior to that of vincristine, a commonly used chemotherapy drug, highlighting the drug's advantage in correcting the abnormalities of characteristic molecules of the disease. (6) This study utilizes IGH::DUX4 A PDX animal model was constructed using primary cells from leukemia patients to evaluate the drug's efficacy and safety in vivo. Animal experiments confirmed that iP300w effectively reduced the proportion of human leukemia cells in the peripheral blood of model mice and significantly improved splenomegaly caused by leukemia. The spleen weight in the treated group was significantly lower than that in the control group. No significant abnormal changes in mouse body weight occurred during the experimental period, demonstrating that the dosing regimen has a preliminary safety range. Further pathological examination of spleen tissue sections showed that iP300w significantly alleviated diffuse infiltration of leukemia cells, and the damaged spleen tissue structure was partially repaired, comprehensively validating the drug's therapeutic effect and safety in vivo. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the DUX4 / IGH sequence constructed onto the commercial empty vector pACT-vector (blue area), with the restriction enzyme sites being BamHI and NotHI.

[0022] Figure 2 This is a schematic diagram showing the EP300, CREBBP, TAZ1, TAZ2, BROMO, and TCF12 sequences constructed onto the commercial empty vector pBIND-vector (the blue part), with the restriction enzyme sites being BamHⅠ and XbaⅠ.

[0023] Figure 3 This is a schematic diagram of shRNA constructed onto the commercial lentiviral vector pLV-CMV-mcherry-puro-H1 (the blue area), with the restriction enzyme sites being BamHI and EcoRI.

[0024] Figure 4 yes IGH::DUX4 Mammalian cell hybridization assays of the fusion protein and CBP / p300 protein. Plasmids pACT-DUX4 / IGH, pBIND-TCF12 (positive control), pBIND-EP300, pBIND-CREBBP, pBIND-TAZ1, pBIND-TAZ2, and pBIND-BROMO were constructed and transfected into 293T cells. Dual-luciferase assays were performed 48 hours after transfection. Each experiment was performed in triplicate. P <0.05 indicates a significant difference.

[0025] Figure 5 It is CBP / p300 in IGH::DUX4 Knockout experiments in type II leukemia cells. Figure 5 A-5B is the knockdown efficiency detection for EP300 / CREBBP; Figure 5 C-5E are cut versions of ERGalt ( Figure 5 C), CLEC12Aalt ( Figure 5 D), c6orf89alt ( Figure 5 Detection of E) expression levels; Figure 5 F-5G is IGH::DUX4 Downstream target gene AGAP1 ( Figure 5 F) and TCF12 ( Figure 5 Expression levels of G were detected. shScramble served as the control group, while shEP300 and shCREBBP were used in the laboratory group, with three different knockdown plasmids designed for each knockdown. Each experiment was performed in triplicate. P <0.05 indicates a significant difference.

[0026] Figure 6 It is a drug sensitivity assay for different subtypes of acute B-lymphoblastic leukemia cells. Figure 6 A is the analysis of cell viability of different subtypes of acute B-lymphoblastic leukemia after treatment with different concentrations of iP300w; Figure 6 B represents the cell viability analysis of different subtypes of acute B-lymphoblastic leukemia cells after treatment with different concentrations of iP300w for 24 hours. Figure 6 C represents the cell viability analysis of different subtypes of acute B-lymphoblastic leukemia (ABL-100) cells after treatment with different concentrations of iP300w for 48 hours. NALM6, REH, SEM, and SUPB15 represent four different subtypes of ABL-100 cells, with NALM6 being the most significant. IGH::DUX4 Fusion precursor B-cell leukemia cells. Each experiment was biologically replicated three times. P <0.05 indicates a significant difference.

[0027] Figure 7 The iP300w can improve this. IGH::DUX4 A secondary blow to type 2 leukemia. Figure 7 The A-7C is an iP300w processor that can handle 12 hours of processing time. Figure 7 A) 24 hours ( Figure 7 B) and 48 hours ( Figure 7 C) Following this, the expression level of ERGalt was detected. Each experiment was performed in three biological replicates. P <0.05 indicates a significant difference.

[0028] Figure 8This analysis uses flow cytometry to detect peripheral blood flow in mice under different treatment groups. PBS is phosphate-buffered saline solution, VCR is the chemotherapy drug vincristine, and iP300w is a CBP / p300 inhibitor. Channel BV510-A detects the percentage of human CD45+ cells, while channel FITC-A detects the percentage of mouse CD45+ cells.

[0029] Figure 9 This is an analysis of the spleen and body weight of mice in different treatment groups. Figure 9 A-9B are photographs of the spleen from different treatment groups. Figure 9 A) and size statistics ( Figure 9 B); Figure 9 C represents the body weight of mice in different treatment groups. PBS is phosphate-buffered saline solution, VCR is the chemotherapy drug vincristine, and iP300w is a CBP / p300 inhibitor.

[0030] Figure 10 These are pathological sections of the spleen from mice in different treatment groups. PBS is phosphate-buffered saline solution, VCR is the chemotherapy drug vincristine, and iP300w is a CBP / p300 inhibitor. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all reagents used in the embodiments are commercially available analytical grade, and all experimental methods used are conventional methods.

[0032] Terminology Explanation Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0033] In this disclosure, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0034] In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0035] In this disclosure, unless otherwise specified, the term " IGH::DUX4 "Gene rearrangement" is abbreviated as DUX4-r, specifically referring to chromosome 4. DUX4 Fragment inserted into chromosome 14 IGH Gene rearrangement formed by a gene locus; tumors carrying this molecular characteristic are uniformly classified as DUX4 rearranged B lymphoblastic leukemia / lymphoma, which is divided into two clinical phenotypes: leukemia and lymphoma.

[0036] In this disclosure, unless otherwise specified, the term " REH "yes ETV6-RUNX1 (TEL-AML1) Fusion positive subtype, " SEM "yes KMT2A-AFF1 (MLL-AF4, t (4;11)) Integrating Pro-B ALL subtype, " SUP-B15 "yes BCR-ABL1 p190 (Ph + ） B-ALL subtypes are three classic B-ALL molecular subtypes, all of which are non-B-ALL. IGH::DUX4 Rearrangement type.

[0037] In this disclosure, unless otherwise specified, the term "NALM6" refers to the classic pattern cell that is IGH-DUX4 fusion positive, belonging to the WHO-specific DUX4 rearrangement B-ALL. This subtype is characterized by: IGH enhancer-driven abnormal overexpression of truncated DUX4, suppressing wild-type ERG; characteristic immunomarkers: CD371 (GPR56) positivity, often accompanied by weak CD2 positivity; predominantly affects adolescents / young adults (AYA), with an intermediate prognosis; NALM-6 is used as the standard cell model in numerous studies on the DUX4 pathway, 3D genomics, and allele silencing.

[0038] In this disclosure, unless otherwise specified, the terms "pharmaceutical composition" or "composition" may refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active ingredient iP300w with liquid excipients, finely chopped solid excipients, or both.

[0039] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0040] In this disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients that are incompatible with the iP300w of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0041] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0042] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available. The main experimental materials are shown in Table 1.

[0043] Table 1. Main Experimental Materials Unless otherwise stated, this disclosure uses various sequences as shown in Table 2.

[0044] Table 2. Various sequences Note: The underlined parts are homologous arm sequences. Declaration of Experimental Materials: The empty vectors used in this disclosure (hereinafter referred to as: pACT-vector, pBIND-vector, pG5-Luciferase and pLV-CMV-mcherry-puro-H1) were all preserved in our laboratory and used to verify the expression regulation and function of the target gene.

[0045] It should be clearly stated that the specific construction method of the aforementioned carrier is not an improvement of this invention, nor does this invention rely on its specific properties. Those skilled in the art can purchase the basic carrier commercially and, based on methods known in the art, obtain an equivalent tool with the same function without any inventive effort.

[0046] The technical solution and its effects of this invention have been verified on multiple independent models, and the verification results show a consistent trend. Therefore, the screening method and the biological function of the obtained genes described in this application do not depend on individual differences of a specific vector and have universal applicability.

[0047] I. Experimental Methods 1. Cell culture: HEK293T, NALM6, REH, SEM, and SUPB15 cells were obtained from the laboratory cell bank of the Shanghai Institute of Hematology. STR identification and mycoplasma testing were performed before the experiments. HEK293T cells were preferably cultured in DMEM complete medium; NALM6, REH, SEM, and SUPB15 cells were preferably cultured in RPMI-1640 complete medium. The complete medium contained 10% fetal bovine serum and 1% penicillin-streptomycin, and the culture conditions were 37 ℃, 5% CO2, and saturated humidity. Suspension cells were maintained in the logarithmic growth phase and passaged at a ratio of approximately 1:3; adherent HEK293T cells were passaged at a ratio of approximately 1:7.

[0048] 2. Compounds and plasmids: The iP300w was prepared by Sigma according to the publicly available synthetic route. Its purity was confirmed by HPLC before use, and a stock solution was prepared with DMSO.

[0049] The plasmid construction details are as follows: (1) The DUX4 / IGH sequence was constructed into the empty pACT-vector (blue part), and the restriction sites were BamHI and NotHI (e.g., ...). Figure 1 (As shown).

[0050] (2) The sequences EP300, CREBBP, TAZ1, TAZ2, BROMO, and TCF12 were constructed into the commercial empty vector pBIND-vector (blue part), and the restriction enzyme sites were BamHⅠ and XbaⅠ (e.g., Figure 2 (As shown).

[0051] (3) shRNA was constructed into the commercial lentiviral vector pLV-CMV-mcherry-puro-H1 (blue portion), with restriction enzyme sites of BamHI and EcoRI (e.g., ...). Figure 3As shown in the table below, the constructed shRNA sequences are referenced.

[0052] The four empty vector plasmids used in this experiment—pACT-vector, pBIND-vector, pG5-Luciferase, and pLV-CMV-mcherry-puro-H1—were all preserved in the laboratory. Homologous recombination was used to construct various recombinant vectors: specific PCR primers containing homologous arms were designed based on the target gene sequence to amplify the target gene fragment (see SEQ ID NO:1-SEQ ID NO:7 in Table 2). After purifying the target fragment using a PCR product recovery kit, the purified fragment was ligated into a linearized vector via homologous recombination. The ligation product was transformed into DH5α competent cells, plated on corresponding antibiotic selection plates for single-clone screening, and single-clone colonies were picked for sequencing identification. Positive clones with correct sequencing results were selected for amplification culture. Plasmids were extracted using an endotoxin-free plasmid extraction kit, and plasmid concentration was detected using NanoDrop 2000. Qualified plasmids were stored at -20℃ for later use.

[0053] The plasmids used in this study's mammalian two-hybrid experiments included recombinant plasmids pACT-DUX4 / IGH, pBIND-EP300, pBIND-CREBBP, pBIND-TAZ1, pBIND-TAZ2, pBIND-BROMO, and pBIND-TCF12, as well as the pG5-Luciferase reporter plasmid. For cell knockdown experiments, pLV-CMV-mcherry-puro-H1 was used as the backbone vector to construct shRNA interference vectors targeting the EP300 and CREBBP genes. A disordered negative control vector, shScramble, was also included. Three specific shRNA interference sequences were designed for each of the two target genes, EP300 and CREBBP. All six shRNA nucleotide sequences and the control sequence are detailed in Table 2 (SEQ ID NO:8-SEQ ID NO:21). All constructed recombinant knockdown vectors were sequenced and verified; the inserted fragment sequences were completely consistent with the designed sequences and could be stably used in subsequent cell gene knockdown functional experiments.

[0054] 3. Two-hybrid experiments in mammalian cells: 24 hours before transfection, HEK293T cells were seeded into 48-well cell culture plates at a seeding density of 1 × 10⁶ cells per well. 5Cells were collected. When the cell confluence reached approximately 80%, the pACT-DUX4 / IGH plasmid was co-transfected with pBIND-EP300, pBIND-CREBBP, and truncated plasmids of each domain, respectively, and simultaneously co-transfected with the pG5-Luciferase reporter plasmid. Forty-eight hours after transfection, the intracellular firefly luciferase activity and internal control luciferase activity were measured, and the relative luciferase activity values ​​were used to assess the strength of protein-protein interactions. Each experiment was performed in at least three technical replicates, and the entire experiment was independently repeated three times to ensure the reliability of the results.

[0055] 4. Cell knockdown experiment: HEK293T cells were transfected with shEP300, shCREBBP interference plasmids, and shScramble negative control plasmid via lentiviral packaging. Cell supernatants were collected to obtain the corresponding lentiviral solutions. NALM6 cells were then infected with the prepared lentiviral solutions, and the cell infection positivity rate was assessed 96 hours after infection. Cells with an infection positivity rate of 80% or higher were directly harvested for subsequent molecular biology experiments. Cells with an infection positivity rate below 80% required flow cytometry sorting or drug screening to enrich positive cells. Qualified cells were collected, and total RNA was extracted. cDNA was obtained by reverse transcription and stored at -20℃ for later use.

[0056] 5. Cell viability and drug efficacy experiments: Four cell lines, NALM6, REH, SEM, and SUPB15, were selected and cultured at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mg / L in 96-well cell culture plates and treated with gradient concentrations of the small molecule compound iP300w for 24 h, 48 h, or 72 h, respectively. Cell viability was determined using the CCK-8 cell viability assay, and the half-maximal inhibitory concentration (IC50) of iP300w for each cell type was calculated using nonlinear regression. 50 In addition, ICs are used. 50 NALM6 cells were treated with iP300w at a concentration of 10 ...

[0057] 6. RT-qPCR experiment: The cDNA obtained from reverse transcription of the cells in each group was used as an amplification template for RT-qPCR quantitative detection. The specific primer sequences for all target genes and internal reference genes in this experiment are detailed in Table 2 (SEQ ID NO:22-SEQ ID NO:37). The RT-qPCR amplification procedure was strictly followed according to the instructions of the fluorescent quantitative enzyme used; the total volume of the standard reaction system was 10 μL, with the following components: 3.5 μL sterile deionized water, 0.25 μL upstream primer, 0.25 μL downstream primer, 5 μL SYBR Green fluorescent quantitative premix, and 1 μL cDNA template. β-actin was selected as the internal reference gene for gene expression data normalization. Three biological replicates were performed for each sample group to ensure accurate and reliable quantitative results.

[0058] 7. Drug administration experiments in PDX animal models: Four-week-old female NSG immunodeficient mice (approximately 10–14 g in weight) were housed in an SPF-grade animal facility at a temperature of 20–26°C, humidity of 40%–70%, with a 12-hour light-dark cycle and free access to food and water. A PDX model was established by intravenous injection of patient-derived DUX4 / IGH rearranged leukemia cells. After successful modeling, the mice were randomly divided into three groups (n=10 per group) and administered PBS, vincristine, or iP300w via intraperitoneal injection, respectively, for five days a week for four weeks. After the treatment period, the efficacy and preliminary safety were evaluated by peripheral blood flow cytometry analysis of the proportion of human CD45+ cells, spleen weight, body weight changes, and spleen pathological sections. The animal experiments were approved by the Shanghai Nanmo Animal Ethics Committee and conducted in accordance with ethical approvals and animal welfare regulations.

[0059] 8. Statistical Standards: Data are expressed as mean ± SD; two-tailed Student's t-tests were used for comparisons between two groups, and one-way ANOVA with multiple comparison corrections was used for comparisons among multiple groups; IC 50 The calculation was performed using a four-parameter logistic curve fitting method. P A value <0.05 is considered statistically significant. Unless otherwise specified, cell experiments should be performed at least three independent biological replicates.

[0060] II. Experimental Results 1. IGH::DUX4 It can interact with CBP / p300 Analysis using a mammalian cell double-hybrid assay revealed... IGH::DUX4 The fusion protein interacts with both EP300 (p300) and CREBBP (CBP) proteins. Further truncation analysis of different domains of p300 / CBP revealed that domains TAZ1 and TAZ2 interact with... IGH::DUX4The fusion proteins exhibit significant interactions, with TAZ1 showing the strongest interaction ability. Figure 4 This illustrates IGH::DUX4 It can interact with CBP / p300.

[0061] 2. EP300 / CREBBP participated in IGH::DUX4 Mediated oncogenic splicing and transcriptional activation processes Through IGH::DUX4 Knock down the expression of EP300 or CREBBP in type 2 leukemia cells ( Figure 5 (A-5B), RT-qPCR analysis revealed that with the knockdown of EP300 or CREBBP, and... IGH::DUX4 Type 2 leukemia-related cutoffs were downregulated. Figure 5 C-5E), and IGH::DUX4 The expression levels of downstream target genes also decreased significantly. Figure 5 (F-5G), indicating that the EP300 / CREBBP was indeed involved. IGH::DUX4 Mediated oncogenic splicing and transcriptional activation processes.

[0062] 3. The iP300w has a subtype selective killing effect. Cell viability assays revealed that iP300w treatment only affected cells with good cell viability. IGH::DUX4 Type I acute B-lymphoblastic leukemia cells have a killing effect ( Figure 6 A), and the lethal effect is time- and dose-dependent ( Figure 6 (B-6C). This indicates that the iP300w has a subtype-selective killing effect.

[0063] 4. iP300w significantly reduced ERGalt expression levels. RT-qPCR analysis revealed that IC 50 NALM6 cells were treated with iP300w at a concentration 12 hours later ( Figure 7 A) 24 hours ( Figure 7 B) or 48 hours ( Figure 7 C), all of which can significantly reduce the expression level of ERGalt. ERGalt is IGH::DUX4 In the treatment of type II leukemia, iP300w significantly suppressed ERGalt expression levels, indicating that iP300w has potential for secondary impact events. IGH::DUX4 Treatment efficacy for type 2 leukemia.

[0064] 5. The iP300w has an inhibitory effect on leukemia. Flow cytometry analysis revealed that, compared with the PBS control group, iP300w administration significantly reduced the percentage of leukemia cells in the peripheral blood of mice. Figure 8This indicates that the iP300w has a good inhibitory effect on leukemia.

[0065] Dissection of the spleens of mice in different treatment groups revealed that, compared with the PBS control group, the spleens of mice treated with iP300w were significantly smaller. Figure 9 (A-9B), and compared with the PBS control, the mice's body weight did not change significantly ( Figure 9 C), indicating that the iP300w has a good therapeutic effect on leukemia.

[0066] Observation of spleen pathological sections from mice in different treatment groups revealed that ( Figure 10 Compared with the untreated PBS group, iP300w administration reduced diffuse leukemia cell infiltration in the spleen of mice, partially restored the basic structure of the spleen, and showed more significant changes in red pulp reactivity. This indicates that iP300w can improve... IGH::DUX4 Type II leukemia onset.

[0067] The above specific embodiments are merely illustrative of the content of this disclosure and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A p300 / CBP inhibitor in the preparation of a treatment IGH::DUX4 Application in drugs for rearranged leukemia.

2. The application according to claim 1, characterized in that: The p300 / CBP inhibitor is a HAT catalytic domain inhibitor; optionally, the HAT catalytic domain inhibitor is a selective small molecule inhibitor; optionally, the selective small molecule inhibitor is iP300w.

3. The application according to claim 2, characterized in that: IGH::DUX4 The fusion protein lacks the wild-type DUX4 C-terminal activation domain and functionally interacts with the TAZ1 and / or TAZ2 domains of the CBP / p300 protein in a non-C-terminal dependent manner. The iP300w blocks... IGH::DUX4 The interaction between the fusion protein and p300 / CBP inhibits... IGH:: DUX4 Aberrant transcriptional programs mediated by fusion proteins.

4. The application according to claim 3, characterized in that: The iP300w can suppress p300 / CBP-mediated [problems]. IGH::DUX4 The expression of downstream target genes of the fusion protein is inhibited. IGH::DUX4 Fusion protein-induced oncogenic RNA alternative splicing.

5. The application according to claim 4, characterized in that: The oncogenic RNA alternative splice includes the ERGalt aberrant splice variant, and the iP300w can significantly reduce the mRNA expression of ERGalt, and its inhibitory effect on ERGalt is better than that of vincristine.

6. The application according to claim 1, characterized in that: The drug acts on IGH::DUX4 In a PDX model derived from patients with rearranged acute B-lymphoblastic leukemia, it can reduce the proportion of human leukemia cells in the peripheral blood of model mice, alleviate leukemia-induced splenomegaly, and relieve diffuse infiltration of leukemia cells in the spleen and repair spleen tissue structure.

7. The application according to claim 1, characterized in that: The active ingredient of the drug is iP300 or a pharmaceutically acceptable salt thereof; optionally, the drug further includes a pharmaceutically acceptable carrier or excipient; optionally, the drug is an injectable formulation.

8. The application according to claim 1, characterized in that: The drug may be used alone or in combination with other drugs.

9. A treatment IGH::DUX4 A pharmaceutical composition for rearranged acute B-lymphoblastic leukemia, characterized in that: This includes therapeutically effective amounts of iP300w or its pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers or excipients.

10. An in vitro screening method IGH::DUX4 The method for targeting inhibitors of rearranged acute B-lymphoblastic leukemia is characterized by, Includes the following steps: (1) Constructing expressions IGH::DUX4 The precursor B lymphocyte model of the fusion protein was set up without... DUX4 Rearranged B-ALL cells served as a negative control; (2) After co-incubating the candidate compound with the cells in (1), the expression levels of downstream target genes of DUX4, the abundance of ERGalt splicing genes, and other parameters were detected. IGH::DUX4 The binding affinity of the fusion protein to CBP / p300; (3) Select compounds that meet at least one of the following effects as candidate drugs: reduce IGH::DUX4 The fusion protein exhibits strong binding affinity to CBP / p300, downregulates ERGalt splice abundance, and modulates the expression levels of downstream target genes of DUX4; the candidate drug can specifically inhibit... IGH::DUX4 Positive cell proliferation indicates the presence of the target compound.

11. The method according to claim 10, characterized in that: In step (2), the protein interaction detection method is selected from at least one of Co-IP, FRET, and PLA (Parallel Linkage Assay); the gene and splice assay is performed using at least one of RT-qPCR, RNA sequencing, and Western blot (WB).

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