Use of dnttip1 gene in preparation of acute leukemia treatment drugs

Abstract

The application of DNTTIP1 gene in preparing acute leukemia treatment drugs belongs to the technical field of biological medicine, and provides a new solution to the preparation of acute leukemia treatment drugs. The present application uses DNTTIP1 gene as a biomarker drug target, and combines the inhibitor MS-275 of the interaction protein HDAC1 of DNTTIP1 and the downstream target gene BMF analog ABT199 to prepare acute leukemia treatment drugs. The deletion of deoxynucleotide transferase terminal interaction protein 1 gene DNTTIP1 will damage the recruitment of histone deacetylase 1 HDAC1 to chromatin, cause high acetylation of histone H3 lysine 27 on B-cell lymphoma 2 modifier factor BMF promoter, and re-activate BMF. The re-activated BMF competitively destroys the BCL2-mediated survival pathway, triggers coordinated autophagy and apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the DNTTIP1 gene in the preparation of drugs for the treatment of acute leukemia. Background Technology

[0002] Acute leukemia (AL) is a malignant clonal disease of hematopoietic stem cells, characterized by the proliferation of abnormal primitive and immature cells in the bone marrow, inhibiting normal hematopoiesis, and potentially infiltrating extramedullary organs such as the liver, spleen, and lymph nodes. Based on the affected cell types, AL is mainly divided into two categories: acute lymphoblastic leukemia and acute myeloid leukemia (AML). With the application of targeted and immunotherapies, leukemia treatment has made progress, but it still ranks sixth in cancer mortality in China. Developing novel targeted drugs and treatment regimens remains crucial for reducing relapse and mortality rates and prolonging patient survival.

[0003] DNTTIP1 has been reported to have potential pro-cancer functions in various solid tumors: in nasopharyngeal carcinoma, DNTTIP1 promotes cancer cell metastasis by recruiting HDAC1 to the promoter of bispecific phosphatase 2 (DUSP2), thereby activating the extracellular signal-regulated kinase (ERK) signaling pathway; in oral squamous cell carcinoma, DNTTIP1 drives tumor progression by promoting HDAC1-mediated deacetylation of the tumor protein p53; in lung cancer, upregulation of DNTTIP1 enhances epithelial-mesenchymal transition and metastasis by recruiting lysine-specific demethylase 1 (LSD1) to the promoter of epithelial cadherin E, inhibiting its transcription. However, in hematologic malignancies, only one study reported that DNTTIP1 overexpression may serve as a specific biomarker for AML through screening of a large database, but its mechanism has not been further explored.

[0004] HDAC inhibitors, such as entenoxetine (MS-275) and chidamide (CI-994), broadly inhibit the activity of HDAC1 / 2 / 3 and other deacetylases, increasing histone acetylation levels and indirectly activating the expression of tumor suppressor genes. However, their clinical application has the following significant limitations: low response rate; off-target effects, non-selective inhibition of multiple histone deacetylase HDAC subtypes (such as HDAC1 / 2 / 3 / 6 / 8) leading to normal cytotoxicity (such as thrombocytopenia and cardiotoxicity); epigenetic heterogeneity, as relying solely on HDAC inhibition cannot precisely regulate specific pro-apoptotic genes; and lack of predictive biomarkers, as the efficacy of existing HDAC inhibitors is not clearly correlated with HDAC expression levels, making it impossible to screen potential beneficiaries in clinical practice.

[0005] BMF belongs to the BH3 family. While its analogues, such as venetoclax (ABT-199), have shown significant efficacy in hematological malignancies, their clinical application still faces key bottlenecks such as high drug resistance, a narrow therapeutic window, and a lack of accurate predictive biomarkers. Resistance mechanisms involve compensated upregulation of BCL-XL / MCL-1 and BCL2 mutations. Some patients exhibit resistance, either primary or secondary to venetoclax; monotherapy is prone to inducing tumor lysis syndrome or myelosuppression. Summary of the Invention

[0006] The problem this invention aims to solve is to provide a new solution for the preparation of drugs for the treatment of acute leukemia, and proposes the application of the DNTTIP1 gene in the preparation of drugs for the treatment of acute leukemia.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An application of the DNTTIP1 gene in the preparation of drugs for the treatment of acute leukemia involves using the DNTTIP1 gene as a biomarker drug target, and combining the inhibitor MS-275 of the DNTTIP1 interacting protein HDAC1 with the downstream target gene BMF mimic ABT199 in the preparation of drugs for the treatment of acute leukemia.

[0009] Furthermore, the DNTTIP1 gene was used as a biomarker drug target, and the HDAC1 inhibitor MS-275 and the pathway protein inhibitor olaparib were combined for use in the preparation of drugs for the treatment of acute leukemia.

[0010] Furthermore, the DNTTIP1 gene was used as a biomarker drug target, and the downstream target gene BMF mimic ABT199 and the pathway protein inhibitor olaparib were combined for use in the preparation of drugs for the treatment of acute leukemia.

[0011] Furthermore, using the DNTTIP1 gene as a biomarker drug target, the HDAC1 inhibitor MS-275, the downstream target gene BMF mimic ABT199, and the pathway protein inhibitor olaparib were combined for the preparation of drugs for the treatment of acute leukemia.

[0012] Furthermore, using the DNTTIP1 gene as a biomarker drug target, the inhibitor of its interacting protein HDAC1, MS-275, is a small molecule compound with the chemical formula C. 21 H 20 N4O3, structural formula is

[0013] .

[0014] Furthermore, the DNTTIP1 gene was used as a drug target for biomarkers, and its downstream target gene BMF mimic, ABT199, is a small molecule compound with the chemical formula C. 45 H 50 ClN7O7S, structural formula is

[0015] .

[0016] Furthermore, the DNTTIP1 gene was used as a biomarker drug target, and its pathway protein inhibitor, olaparib, is a small molecule compound with the chemical formula C. 24 H 23 FN4O3, structural formula is

[0017] .

[0018] Furthermore, the deletion of the deoxynucleotidyl transferase terminal interacting protein 1 gene DNTTIP1 impairs the recruitment of histone deacetylase 1 HDAC1 to chromatin, leading to hyperacetylation of histone H3 lysine 27 on the promoter of B-cell lymphoma 2-modifying factor BMF and reactivating BMF. The reactivated BMF competitively disrupts the BCL2-mediated survival pathway, triggering coordinated autophagy and apoptosis.

[0019] Furthermore, B-cell lymphoma 2-modifying factor (BMF) is a target gene for regulation.

[0020] Furthermore, using the DNTTIP1 gene as a primary biomarker target for drug development, and targeting the DNTTIP1-HDAC1-BMF axis, we will intervene in the HDAC1 and BMF pathways, and combine HDAC1 inhibitors and BMF mimics to verify their synergistic function in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Based on the DNA repair pathways regulated by DNTTIP1, we will add PARP inhibitors to promote the differentiation and apoptosis of acute leukemia cells and reduce DNA damage repair in acute leukemia cells. We will then prepare small molecule drugs or gene inhibitors for use in the development of drugs to treat acute leukemia.

[0021] Furthermore, drugs that target the DNTTIP1 gene include nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, peptides, proteins, or adeno-associated viruses.

[0022] Furthermore, the nucleic acid molecule is an antisense oligonucleotide, double-stranded RNA, small interfering RNA, or short hairpin RNA.

[0023] Furthermore, the adeno-associated virus contains a nucleotide sequence that interferes with the expression of the DNTTIP1 gene.

[0024] Furthermore, the acute leukemia treatment drug also contains pharmaceutically acceptable excipients, which are one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, or water.

[0025] Furthermore, the acute leukemia treatment drug is in the form of tablets, pills, powders, or injections.

[0026] The beneficial effects of this invention are:

[0027] This invention relates to the application of the DNTTIP1 gene in the preparation of drugs for the treatment of acute leukemia, involving deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1), its interacting protein HDAC1, and its downstream target gene BMF. In vivo and in vitro experiments have shown that DNTTIP1 expression levels are associated with the prognosis of acute leukemia. Overexpression of DNTTIP1 promotes the proliferation of acute leukemia cells, while knockdown of DNTTIP1 inhibits the proliferation of acute leukemia cells, promotes their differentiation and apoptosis, and weakens DNA damage repair in acute leukemia cells. DNTTIP1 has the potential to serve as a biomarker for diagnosis, prognosis assessment, and monitoring the progression of acute leukemia. Since there are currently no targeted drugs for DNTTIP1, this invention first targets the DNTTIP1-HDAC1-BMF axis, intervening in the HDAC1 and downstream BMF pathways, combining HDAC1 inhibitors and BMF mimics to verify their synergistic function in AML / ALL, and further exploring the effects of the three-drug combination by adding a PARP inhibitor based on the exploration of downstream pathways regulated by DNTTIP1. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the eight genes that intersect with the HDACs scores obtained from ssGSEA analysis of the STRING protein interaction network of the HDAC family in this invention.

[0029] Figure 2 Schematic diagram of the MiDAC complex;

[0030] Figure 3 A comparison of the protein expression of DNTTIP1 and HDAC1 in primary bone marrow cells from normal individuals and leukemia patients as detected by Western blot assay;

[0031] Figure 4 Pearson correlation plot of DNTTIP1 expression and HDACs;

[0032] Figure 5The results of the analysis of DNTTIP1 in different acute leukemia databases are as follows: (A) is the expression analysis of DNTTIP1 in the acute leukemia GEO dataset GSE48558; (B) is the prognostic analysis of the level of DNTTIP1 expression in the acute lymphoblastic leukemia TARGET database; and (C) is the prognostic analysis of the level of DNTTIP1 expression in the acute myeloid leukemia TCGA database.

[0033] Figure 6 To demonstrate that DNTTIP1 knockdown significantly inhibited cell proliferation in acute leukemia cells RS4;11, (A) shows Western blot validation of two shRNAs (sh1, sh4) targeting DNTTIP1 knockdown, (B) shows qRT-PCR validation, and (C) shows cell counting analysis after DNTTIP1 knockdown.

[0034] Figure 7 To show that DNTTIP1 knockdown significantly reduced colony formation in acute leukemia cells RS4;11, (A) is a representative image showing the significant reduction in clone number after DNTTIP1 knockdown, and (B) is a statistical graph showing the specific clone number in the DNTTIP1 knockdown group compared to the control group.

[0035] Figure 8 The comparison diagram shows the effect of DNTTIP1 knockdown on leukemia cell proliferation in in vivo mouse models at different time points. (A) is 2 weeks, (B) is 3 weeks, (C) is 4 weeks, and (D) is 5 weeks.

[0036] Figure 9 This is a graph of multi-omics sequencing analysis;

[0037] Figure 10 The diagram shows the changes in autophagy and apoptosis-related proteins after DNTTIP1 knockdown as detected by Western blot assay. (A) shows the changes in apoptosis-related proteins after DNTTIP1 knockdown, and (B) shows the changes in autophagy-related proteins after DNTTIP1 knockdown.

[0038] Figure 11 This is a schematic diagram illustrating the influence of downstream target gene BMF on autophagy and apoptosis pathways, where BAX / BAK are pro-apoptotic proteins and Beclin-1 is an autophagy-related protein.

[0039] Figure 12The synergistic inhibition score of ABT199 and MS-275 in combination in acute leukemia is shown in (A), which is the ZIP synergistic score after bone marrow cells of acute myeloid leukemia patients were exposed to multiple concentrations of ABT-199 and MS-275 for 48 hours; and (B) is the ZIP synergistic score after bone marrow cells of acute lymphoblastic leukemia patients were exposed to multiple concentrations of ABT-199 and MS-275 for 48 hours.

[0040] Figure 13 The diagram shows the synergistic inhibitory effect of the combination of ABT-199, MS-275 and Olaparib in acute leukemia cells. (A) is a representative drug synergy matrix diagram of the three-drug combination, and (B) is a statistical graph of the synergistic scores of the two-drug combination and the three-drug combination. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described specific embodiments are merely a part of the embodiments of the invention, and not all of them. The components of the specific embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations, and the invention may also have other embodiments.

[0042] Therefore, the following detailed description of specific embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected specific embodiments of the invention. All other specific embodiments obtained by those skilled in the art based on these specific embodiments without inventive effort are within the scope of protection of this invention.

[0043] To further understand the invention's content, features, and effects, the following specific embodiments are provided, along with accompanying drawings. Figure 1 - Appendix Figure 13 Detailed explanation is as follows:

[0044] Example 1:

[0045] The application of the DNTTIP1 gene in the preparation of drugs for the treatment of acute leukemia, wherein the new target is the deoxynucleotidyl transferase terminal interacting protein 1 gene (DNTTIP1). This invention is the first to conduct a detailed mechanistic exploration and verification of DNTTIP1 in acute leukemia, involving its interacting protein histone deacetylase 1 (HDAC1) and its downstream target gene B-cell lymphoma 2 (BCL2) modifying factor (BMF).

[0046] The loss of DNTTIP1 impairs the recruitment of histone deacetylase 1 (HDAC1) to chromatin, leading to hyperacetylation of histone H3 lysine 27 on the promoter of B-cell lymphoma 2-modifying factor (BMF) and reactivation of BMF. The reactivated BMF competitively disrupts the BCL2-mediated survival pathway, triggering coordinated autophagy and apoptosis.

[0047] Furthermore, B-cell lymphoma 2 modifying factor BMF is a downstream target gene of the regulation.

[0048] Furthermore, DNTTIP1 expression levels are associated with the prognosis of acute leukemia. Overexpression of DNTTIP1 promotes the proliferation of acute leukemia cells, while knockdown of DNTTIP1 inhibits the proliferation of acute leukemia cells and promotes their differentiation and apoptosis.

[0049] Figure 1 This diagram illustrates the eight genes intersecting with HDAC scores obtained through the STRING protein interaction network and ssGSEA analysis of the HDAC family, as shown in this invention. The vertical axis represents the eight enriched genes: NCOR2 (nuclear receptor co-repressor 2), DNTTIP1 (deoxynucleotidyl transferase terminal interacting protein 1), BRMS1 (breast cancer metastasis inhibitor 1), BCL6 (B-cell lymphoma 6), H2AX (histone H2A family member X), MEF2D (myotrophic factor 2D), HSF1 (heat shock transcription factor 1), and NADSYN1 (NAD synthase 1). The horizontal axis represents the 18 subtypes of the HDAC family, including HDACs (histone deacetylases) and SIRT (silencing information regulators); these eight genes are the most closely related to HDACs in acute leukemia. Previous comprehensive genome annotation has identified 18 types of human deacetylases (HDACs) across four classes, and previous studies have repeatedly demonstrated that class I HDACs are abnormally expressed in neoplastic diseases compared to other HDACs. Furthermore, the top 100 proteins interacting with these 18 human deacetylases were screened using STRING protein interactions. Further, comprehensive analysis of TCGA transcriptomics data (using ssGSEA analysis) identified 1649 HDACs-related genes through HDACs scoring. The STRING protein interaction network and the genes screened using HDACs scoring were then enriched as follows: Figure 1 As shown, eight genes most closely related to HDACs in acute leukemia were identified. Among them, DNTTIP1 showed unique binding specificity to HDAC1 and HDAC2 of the class I HDAC family, thus distinguishing DNTTIP1 from the other seven genes and demonstrating the close relationship between DNTTIP1 and the class I family of HDACs in acute leukemia.

[0050] Figure 2 This is a schematic diagram of the MiDAC complex. MIDEAS represents DNA damage checkpoint mediators, and AC represents acetyl groups. DNTTIP1 is one of the eight major HDAC complexes, the Mitosis-associated deacetylation complex (MiDAC), a core scaffold protein essential for assembly and stability. Unlike other HDAC complexes, MiDAC exhibits a unique structural organization, such as... Figure 2 As shown, DNTTIP1 and the mitotic deacetylase-associated SANT domain protein (MIDEAS) form the core scaffold, recruiting four HDAC1 / 2 subunits, making it the complex with the highest HDAC subunit content among all HDAC complexes to date. Notably, the outward arrangement of the HDAC1 / 2 subunits suggests a unique regulatory mechanism that may allow simultaneous binding to multiple nucleosomes to enhance deacetylase activity across chromatin domains.

[0051] Figure 3This image shows a comparison of DNTTIP1 and HDAC1 protein expression in primary bone marrow cells from healthy individuals and leukemia patients, as detected by Western blot analysis. AML represents acute myeloid leukemia, ALL represents acute lymphoblastic leukemia, D represents healthy donors (D1: donor 1, D2: donor 2), P represents leukemia patients (P1: AML patient 1, P2: AML patient 2, P3: AML patient 3, P4: ALL patient 4), and TUBULIN represents microtubules, a common internal control protein. The experimental procedure involved collecting bone marrow samples from newly diagnosed acute leukemia (AML / ALL) patients and healthy donors at the First Affiliated Hospital of Harbin Medical University. This experiment was approved by the Medical Ethics Committee of the First Affiliated Hospital of Harbin Medical University, and informed consent was obtained. Mononuclear cells (MNCs) were isolated from the bone marrow samples using Ficoll density gradient centrifugation (TBD Sciences, Tianjin). Cell lysis was performed using RIPA lysis buffer (Beyotime #P0013B) containing a protease inhibitor (Proteintech #PR20016), and protein concentration was determined using a BCA kit (Beyotime #P0009). 20 μg of protein per well was separated by 10% or 12.5% ​​SDS-PAGE and transferred to a PVDF membrane (Millipore #3010040001). The membrane was blocked with 5% skim milk for 1 hour, incubated overnight at 4°C with primary antibody, washed with PBST, and incubated at room temperature for 2 hours with HRP-labeled secondary antibody. β-tubulin or H3 was used as an internal control, and detection was performed using Super ECL Plus chromogenic buffer (Epizyme #SQ201L). Signal acquisition was performed using a chemiluminescence imaging system (Tanon #5200). Given the potential key oncogenic role of HDAC1 and DNTTIP1 in acute leukemia, the expression levels of HDAC1 and DNTTIP1 in mononuclear cells extracted from bone marrow blood of healthy donors and patients with acute leukemia were further analyzed. Figure 3 The expression levels of HDAC1 and DNTTIP1 in patients with acute leukemia were significantly higher than those in healthy donors, suggesting a possible pro-cancer role of DNTTIP1 in acute leukemia.

[0052] Figure 4 Pearson correlation analysis of DNTTIP1 expression and HDACs shows that DNTTIP1 expression is positively correlated with HDACs scores, and DNTTIP1 is most strongly associated with poor prognosis in acute leukemia, reinforcing its role as an independent prognostic biomarker and a co-regulatory factor of selective HDAC1 / 2 in hematologic malignancies.

[0053] Figure 5The results of the analysis of DNTTIP1 in acute leukemia databases are presented. In the GEO dataset GSE48558, DNTTIP1 expression in acute leukemia samples (including ALL and AML) was significantly higher than that in normal controls, further demonstrating the potential oncogenic role of DNTTIP1. Analysis of the TARGET and TCGA acute leukemia datasets showed that high DNTTIP1 expression levels were associated with significantly shorter overall survival in acute leukemia (AML / ALL), indicating that DNTTIP1 is associated with poor prognosis in acute leukemia and supporting its potential as a prognostic biomarker for hematological malignancies.

[0054] To investigate the functional role of DNTTIP1 in acute leukemia, two independent shRNAs (sh1 and sh4) were designed, each targeting a different region (3'UTR) of DNTTIP1. Their knockdown efficiency was validated in various acute leukemia cell lines, including the B-cell acute lymphoblastic leukemia (B-ALL) cell line RS4;11. Specific target information for DNTTIP1 knockdown is shown in Table 1.

[0055] Table 1

[0056]

[0057] Figure 6 To validate the results of Western blot and qRT-PCR after knocking down DNTTIP1 in acute leukemia cells RS4;11, the experimental procedure was as follows: Human leukemia cell line (RS4;11) was purchased from Cybiocon (Shanghai, China) and cultured in rpm-1640 medium (VivaCell, China) supplemented with 10% fetal bovine serum (FBS) (Vazyme, USA) and 1% penicillin / streptomycin (NCMBiotech, China). All cells were cultured at 37℃ and 5% CO2, and mycoplasma contamination was checked periodically. RNA was extracted using an RNA purification kit (TransGen Biotech, #ER101-01). 1 μg of purified RNA was reverse transcribed using cDNA Synthesis SuperMix (YEASON, #11141ES60). qRT-PCR was performed on a BIOER real-time PCR system using the universal SYBR Green Master Mix (YEASON, #11184ES08). Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene, the relative expression levels of the target gene were calculated using the 2-ΔΔCt method. Figure 6 The absence of DNTTIP1 significantly inhibited the in vitro proliferation of acute leukemia cells.

[0058] Figure 7To investigate the effect of DNTTIP1 knockdown on colony formation in acute leukemia cells (RS4;11), the colony formation assay was performed in acute leukemia cells as follows: 5 × 10³ cells were seeded three times in 6-well plates. The upper layer consisted of 1.5 ml of medium containing 10% serum and 0.36% agar, and the lower layer consisted of 2 ml of the same medium containing 0.75% agar. Cells were cultured in a humidified incubator at 37°C with 5% CO2 for 2–3 weeks. After the incubation period, surviving colonies were stained with iodine chloride nitrosyltetrazole (#V900870, Sigma-Aldrich, Shanghai, China) for 24 hours. ImageJ software was used to image and quantify the stained colonies to assess their viability and quantity. Figure 7 The results showed that DNTTIP1 knockdown significantly reduced the colony-forming ability of these cells.

[0059] Figure 8 Comparison of DNTTIP1 knockdown inhibiting leukemia cell proliferation in in vivo mouse models at different time points; The purpose of the experiment was to further verify the function of DNTTIP1 in acute leukemia. An in vivo RS4;11 acute leukemia xenograft mouse model was constructed. Establishment of cell line-derived xenograft model (CDX): 6-week-old female NCG mice (Jicui Yaokang, Nanjing) were acclimatized for 7 days and then injected with 5×10⁻⁶ dredged ... 6 Luciferase-labeled RS4;11 control (pLKO) or DNTTIP1 knockout (sh4) cells. Two weeks after transplantation, D-luciferin (150 mg / kg, GoldBio #18567-5MG) was injected intraperitoneally, and bioluminescence signals were collected 15 minutes later using IndiGo software (Berthold), monitored weekly. Figure 8 In a mouse in vivo model, shRNA-mediated downregulation of the DNTTIP1 gene significantly inhibited leukemia cell growth, prolonged survival time, and reduced extramedullary infiltration. The consistent reproduction of these phenotypes in both in vitro and in vivo models supports the role of DNTTIP1 as a key driver of acute leukemia development.

[0060] Figure 9 This is a multi-omics sequencing analysis diagram; to explore the functional targets of DNTTIP1, RNA-seq, CUT&Tag, and ATAC-seq sequencing were performed.

[0061] RNA-seq and data analysis: After quality control, RNA from three biological repeats was used to construct libraries using TruSeq PE ClusterKit v3-cBot-HS (Illumina), and 150 bp paired-end sequencing was performed on the NovaSeq platform. Differential expression analysis was performed using DESeq2 (v1.20.0), with a threshold set at FDR ≤ 0.05 (Benjamini-Hochberg correction).

[0062] CUT & Tag and Data Analysis: Using the Hyperactive CUT & Tag Kit (Vazyme #TD904), 10 5 After cells bind to ConA magnetic beads, they are incubated overnight at 4°C with primary antibody (HA, H3K27ac or IgG control), and then bound with secondary antibody and pA-Tn5 transposase complex (1 hour).

[0063] ATAC-seq and Data Analysis: ATAC-seq was performed using the Library Prep Kit (Vazyme, #TD711). 1×10 5 Fresh cells were washed with ice-cold TW buffer and lysed to release the nuclei. After centrifugation (500×g, 5 min, 4°C), the nuclei were resuspended in a transposition mixture (37°C, 30 min) while simultaneously lysing and labeling. The transposed DNA was purified using magnetic DNA extraction beads, amplified by PCR with indexed primers, and processed into sequencing libraries using the TruePrep Index Kit V2 for Illumina (Vazyme, #TD202).

[0064] DNA fragmentation was followed by library construction and sequencing on the Illumina platform. Raw data were quality controlled using FastP (v0.22.0), peak values ​​(q<0.05) were retrieved using MACS3 (v3.0.0a7), and visualization was performed using DeepTools (v3.5.1). IGV was used to display the results. ATAC-seq and data analysis: The Hyperactive ATAC-seq kit (Vazyme #TD711) was used. 5 After cell lysis, the nuclei were transposed via Tn5 (37℃ for 30 minutes), and the DNA was purified and amplified by PCR for 12 cycles. The data analysis workflow was the same as CUT&Tag.

[0065] Enrichment analysis of overlapping genes identified from multi-omics sequencing co-analysis revealed significant enrichment in cancer-related pathways, particularly specific enrichment in autophagy and apoptosis pathways, with significant contributions only from BH3-only family members (BMF, PUMA, NOXA).

[0066] Figure 10 This image shows the changes in autophagy and apoptosis-related proteins detected by Western blot analysis after DNTTIP1 knockdown. PARP (poly(ADP-ribose) polymerase C), cleaved PARP (cleaved poly(ADP-ribose) polymerase C), Caspase 9 (cleaved caspase 9), Caspase 3 (cleaved caspase 3), and cleaved caspase 3 (cleaved caspase 3) are important protein markers of the apoptosis pathway. P62 (autophagy aptamer protein P62) and LC3B (microtubule-associated protein 1 light chain 3B) are also important protein markers of the autophagy pathway. Western blot analysis showed that the changes in apoptosis and autophagy-related proteins demonstrated that DNTTIP1 knockdown in acute leukemia cells activated autophagy and apoptosis pathways, supporting the link between DNTTIP1 and these programmed cell death pathways. The specific experimental procedure for Western blot was as follows: Total cell lysates were prepared using RIPA lysis buffer (Beyotime, #P0013B) and a mixture of protease inhibitors (Proteintech, #PR20016). Protein concentration was detected using an enhanced BCA protein assay kit (Beyotime, #P0009). Equal volumes of protein were dissolved on 10% or 12.5% ​​SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, #3010040001). The membranes were blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation with primary antibody at 4°C. After washing three times with 1×PBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 hours. β-tubulin and H3 were used as loading controls. Finally, the protein bands were visualized using the Super ECL Plus assay kit (Epizyme, #SQ201L). Protein expression levels were visualized using a chemiluminescence imaging system (Tanon, #5200).

[0067] Figure 11 This is a schematic diagram illustrating the influence of downstream gene BMF on autophagy and apoptosis pathways. BMF, as a member of the BCL2 family, participates in both pro-apoptotic and autophagy pathways. BMF competitively binds to BCL-2, thereby releasing the pro-apoptotic proteins BAX / BAK and the key autophagy protein Beclin-1, thus promoting apoptosis and autophagy. Considering the crucial roles of autophagy and apoptosis in cancer treatment, targeting the DNTTIP1-HDAC1-BMF axis can regulate these two cell death pathways, demonstrating significant therapeutic potential.

[0068] Example 2:

[0069] This invention relates to the application of DNTTIP1 as a target in the preparation of therapeutic agents for acute leukemia. It utilizes the DNTTIP1 gene as a novel target for drug development in acute leukemia. Previously, the mechanism of DNTTIP1 in acute leukemia was not clearly explored, and there were no drugs specifically targeting DNTTIP1. This invention is the first to validate the intervention of the DNTTIP1-HDAC1-BMF axis in acute leukemia, thus demonstrating the exploratory potential of DNTTIP1 as a novel target. By intervening in the HDAC1 and BMF pathways, the synergistic function of the combined HDAC1 inhibitor and BMF mimicry was verified in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Furthermore, based on the enrichment of DNA repair-related pathways in the DNTTIP1 regulatory pathway, a PARP inhibitor was added to inhibit the damage repair of acute leukemia cells, thus developing a new therapeutic regimen for acute leukemia.

[0070] Furthermore, the HDAC1 inhibitor is entinolide MS-275, the BMF mimic is venetoc ABT-199, and the PARP inhibitor is olaparib.

[0071] Figure 12 From left to right, the images show ZIP synergistic scores of AML and ALL bone marrow cells after 48 hours of combined treatment with ABT-199 and MS-275 at various concentrations. To further validate the therapeutic potential of two drugs targeting the DNTTIP1-HDAC1-BMF axis—an HDAC1 inhibitor and a BMF mimic—in acute leukemia, a drug synergistic experiment was conducted in primary bone marrow cells from AML and ALL patients. The experimental procedure investigated the efficacy of the combination therapy of the HDAC1 inhibitor MS-275 and the BMF mimic ABT-199. The specific cell viability assay procedure was as follows: Bone marrow cells from AML and ALL patients were seeded in 96-well plates, treated with gradient concentrations of drugs for 48 hours, and viability was detected using a CellTiter-Glo chemiluminescence assay (Promega #G7570). The signals were read using a SpectraMax i3 microplate reader (Molecular Devices), and the cell viability under different drug combinations was analyzed based on the values ​​displayed by the microplate reader. Figure 12As shown, the combination therapy of MS-275 and ABT-199 demonstrated potent synergistic cytotoxicity in primary AML and ALL patient samples, with a synergistic score of 14.943 in acute myeloid leukemia and 13.148 in acute lymphoblastic leukemia, both scores greater than 10, demonstrating the synergistic effect of the two drugs and highlighting their translational potential. Although drug sensitivity varied slightly between patient samples and cell lines, both the HDAC1 inhibitor MS-275 and the BMF mimic ABT-199 effectively inhibited the proliferation of leukemia cells from AML and ALL patients.

[0072] Figure 13 This image shows the combined effects of ABT-199, MS-275, and Olaparib. The aim was to conduct in-depth drug target research targeting the DNTTIP1-HDAC1-BMF axis. The specific experimental procedure was as follows: Acute leukemia cells (RS4;11) were seeded in 96-well plates. The three drugs, ABT-199, MS-275, and Olaparib, were treated with gradient concentrations at both pairwise and triadic combinations for 48 hours. Cell viability was detected using CellTiter-Glo chemiluminescence immunoassay, and signals were read using SpectraMax i3 enzyme-linked immunosorbent assay (ELISA). Cell viability under different drug combinations was analyzed based on the values ​​displayed by the ELISA reader. ZIP synergy scores were used to measure the degree of interaction between drugs, calculated using the SynergyFinder website (https: / / synergyfinder.fimm.fi / ). According to literature reports, a score greater than 10 indicates a synergistic effect.

[0073] Previous studies have reported the function of DNTTIP1 in DNA repair and other pathways, and multi-omics analysis has also found downregulation of DNA repair-related pathways. PARP inhibitors, which are related to DNA repair, have been proven effective in various tumors, including leukemia. Figure 12 It has been demonstrated that the combination of the HDAC1 inhibitor MS-275 and the BMF mimic ABT-199 exhibits potent synergistic toxicity in acute leukemia. This invention incorporates a PARP inhibitor targeting the DNA repair pathway regulated by DNTTIP1, such as... Figure 13 As shown, the synergistic effect of the drugs was demonstrated by the combination of three drugs (PARP inhibitor + HDAC1 inhibitor + BMF mimicry). The synergistic score showed that the synergistic score of any two drug combinations was greater than 10, and the synergistic score of the three-drug combination was the highest. The ZIP synergistic score was greater than 20. This proves that the intervention of genes and pathways regulated by DNTTIP1 clearly inhibits acute leukemia and elucidates the strong potential of targeting the new target DNTTIP1 in the treatment of acute leukemia.

[0074] This invention proposes an innovative solution based on the novel target DNTTIP1 and its regulated HDAC1-BMF mechanism. DNTTIP1, through HDAC1, breaks the epigenetic silencing of BMF, leading to increased BMF expression. Because it is unaffected by genes such as BCL-XL / MCL-1 and BCL2, it may significantly reverse venetoclax resistance. Based on this, this invention proposes the significant therapeutic potential of DNTTIP1 inhibitors (such as gene therapy), and through ample experimental demonstration, shows that when combined with downstream target genes and pathways, the synergistic effect is increased when using a combination of PARP inhibitors, HDAC1 inhibitors, and BMF mimics. This strategy not only overcomes the low response rate of traditional HDAC inhibitors and the resistance limitations of BCL2 inhibitors, but also provides a new direction for combination therapy of leukemia by targeting DNTTIP1, the starting point of the epigenetic regulatory axis.

[0075] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0076] Although this application has been described above with reference to specific embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of this application. In particular, as long as there is no structural conflict, the features in the specific embodiments disclosed in this application can be combined with each other in any way. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, this application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Use of DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia, characterized in that, The application relates to application of DNTTIP1 gene as a biomarker drug target, combination of HDAC1 inhibitor MS-275 and downstream target gene BMF mimic ABT199 in preparation of an acute lymphoblastic leukemia treatment drug.

2. Use of DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia, characterized in that, The application relates to application of DNTTIP1 gene as a biomarker drug target, combination of HDAC1 inhibitor MS-275 and pathway protein inhibitor Olaparib in preparation of an acute lymphoblastic leukemia treatment drug.

3. Use of DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia, characterized in that, The application relates to application of DNTTIP1 gene as a biomarker drug target, combination of downstream target gene BMF mimic ABT199 and pathway protein inhibitor Olaparib in preparation of an acute lymphoblastic leukemia treatment drug.

4. Use of DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia, characterized in that, The application relates to application of DNTTIP1 gene as a biomarker drug target, combination of HDAC1 inhibitor MS-275, downstream target gene BMF mimic ABT199 and pathway protein inhibitor Olaparib in preparation of an acute lymphoblastic leukemia treatment drug. 5.The use of a DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia according to claim 1, characterized in that, The DNTTIP1 gene is used as a biomarker of a drug target, and an inhibitor MS-275 of the interaction protein HDAC1 is a small molecule compound, and the chemical formula is C 21 H 20 N4O3, and the structural formula is 。 6.The use of a DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia according to claim 1, characterized in that, The DNTTIP1 gene is used as a biomarker of a drug target, and the mimic ABT199 of a downstream target gene BMF is a small molecule compound, and its chemical formula is C 45 H 50 ClN7O7S, and the structural formula is 。 7.The use of a DNTTIP1 gene in the preparation of a drug for treating acute lymphoblastic leukemia according to claim 2, characterized in that, The DNTTIP1 gene is used as a biomarker of a drug target, and its pathway protein inhibitor, Olaparib, is a small molecule compound with a chemical formula of C 24 H 23 FN4O3, and a structural formula of 。

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