Method for killing brain tumor cells in vitro

Abstract

Disclosed is a method for killing brain tumor cells in vitro. According to the embodiments of the present invention, the method comprises: bringing isolated brain tumor cells into contact with ABBV-075 to kill brain tumor cells. According to the embodiments of the present invention, the method kills brain tumor cells in vitro more efficiently.

Description

Methods for killing brain tumor cells in vitro Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically, to a method for killing brain tumor cells in vitro. Further, this invention relates to a method for targeting brain tumor cells in vitro, the use of reagents in the preparation of reagent kits, the use of reagents in the preparation of pharmaceuticals, a pharmaceutical composition, a method for inhibiting CREB5 expression, a method for treating brain tumors, and a method for promoting the differentiation of brain tumor cells into astrocytes. Background Technology

[0002] Brain tumors are the most common malignant tumors of the central nervous system, characterized by high invasiveness, high recurrence rate, and treatment resistance. Diffuse intrinsic pontine glioma (DIPG) is a particularly challenging type of brain tumor occurring in the brainstem, typically affecting children and adolescents, and with an extremely poor prognosis. Classified as H3K27M-mutant diffuse midline glioma (DMG-H3K27M), DMG is a newly added brain tumor category in the 2016 WHO classification of malignant tumors of the central nervous system (CNS). DMG occurs in the spinal cord, brainstem, pineal region, and thalamus; its clinical behavior is invasive. The H3K27M mutation is a common gene variant in DIPG and is closely associated with tumor invasiveness and treatment resistance.

[0003] Despite some progress in in vitro killing of brain tumor cells in recent years, current methods for killing brain tumor cells still have many limitations. Current methods for killing brain tumor cells are often accompanied by serious side effects, and their killing effect is limited and their specificity in targeting brain tumor cells is poor.

[0004] Therefore, there is an urgent need to develop a new method for killing brain tumor cells in vitro to improve the efficiency of killing brain tumor cells. Summary of the Invention

[0005] The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides a method for in vitro killing of brain tumor cells.

[0006] This invention is based on the following discoveries of the inventors:

[0007] Current methods for killing brain tumor cells often have serious side effects, low killing efficiency, and poor specificity in targeting brain tumor cells. To overcome this problem, the inventors have proposed a method for killing brain tumor cells in vitro, which improves the efficiency of killing brain tumor cells in vitro and enhances the specificity of targeting brain tumor cells.

[0008] In a first aspect, the present invention provides a method for in vitro killing of brain tumor cells. According to an embodiment of the invention, the method includes: contacting isolated brain tumor cells with ABBV-075 to kill the brain tumor cells. The method according to the embodiment of the invention can improve the efficiency of in vitro killing of brain tumor cells.

[0009] In a second aspect, the present invention provides a method for targeting brain tumor cells in vitro. According to an embodiment of the invention, the method includes: contacting ABBV-075 with a separated cell population, said cell population including brain tumor cells. The method according to the embodiment of the invention can specifically target brain tumor cells in vitro.

[0010] In a third aspect, the invention provides for the use of the reagent in the preparation of a kit. According to embodiments of the invention, the reagent comprises ABBV-075, and the kit is used to inhibit the expression of CREB5 and / or ID1.

[0011] In a fourth aspect, the invention provides for the use of the reagent in the preparation of a medicament. According to an embodiment of the invention, the reagent comprises ABBV-075, and the medicament is used to treat gliomas.

[0012] In a fifth aspect, the present invention provides a pharmaceutical composition for treating or preventing gliomas. According to an embodiment of the invention, the pharmaceutical composition comprises ABBV-075. The pharmaceutical composition according to embodiments of the invention is capable of improving the efficiency of in vitro glioma cell killing and improving the specificity of targeting glioma cells.

[0013] In a sixth aspect, the present invention provides a method for inhibiting CREB5 expression. According to an embodiment of the invention, the method involves contacting ABBV-075 with isolated cells expressing CREB5 to inhibit the expression of said CREB5. The method according to an embodiment of the invention is capable of inhibiting CREB5 expression.

[0014] In a seventh aspect, the present invention provides a method for promoting the differentiation of brain tumor cells into astrocytes. According to an embodiment of the invention, the method includes contacting isolated brain tumor cells with ABBV-075 to promote the differentiation of the brain tumor cells into normal glial cells. The method according to an embodiment of the invention is capable of promoting the differentiation of brain tumor cells into astrocytes.

[0015] In an eighth aspect, the present invention provides a method for treating brain tumors. According to an embodiment of the invention, the method includes administering ABBV-075 to the subject. The method according to an embodiment of the invention is capable of effectively treating brain tumors. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0017] Figure 1 is an RNA-seq data analysis diagram of DIPG treated with the BRD4 inhibitor JQ1 in Example 1.

[0018] Figure 2 shows the qPCR results of JQ1-treated CREB5 and stemness-related genes detected by qPCR in Example 1.

[0019] Figure 3 shows the qPCR results of 18 BRD4 small molecule inhibitors in Example 2.

[0020] Figure 4 shows the cytotoxic effects of five BRD4 small molecule inhibitors on DIPG and PPC cells in Example 2.

[0021] Figure 5 shows the results of the molecular docking experiment between ABBV-075 and JQ1 in Example 3.

[0022] Figure 6 shows the Western blot results of proteins treated with ABBV-075 at different times in Example 3.

[0023] Figure 7 shows the Western blot results of proteins treated with different concentrations of ABBV-075 in Example 3.

[0024] Figure 8 shows the experimental results of downregulating OLIG2 and ID1 protein levels in Example 3 to induce neurite-like differentiation phenotypes in DIPG cells.

[0025] Figure 9 shows the experimental results of the dual-fluorescent reporter system in Example 3, which demonstrates that the super enhancer of CREB5 can significantly promote the promoter activity of CREB5.

[0026] Figure 10 shows the experimental results of the dual-fluorescent reporter system in Example 3, which demonstrates that ABBV-075 can inhibit the expression of CREB5 by suppressing the activity of the super enhancer and promoter of CREB5.

[0027] Figure 11 shows the experimental results of the dual fluorescent reporter system in Example 3, in which 20 nM ABBV-075 significantly inhibited the activity of the CREB5 enhancer.

[0028] Figure 12 shows the growth curves and spheroidization results of ABBV-075 on different DIPG cells in Example 4.

[0029] Figure 13 shows a live fluorescence imaging image from Example 4.

[0030] Figure 14 is a data analysis graph showing the combined therapeutic effect of ABBV-075 and BRM014 on DIPG17 and TT150714 cells in Example 4. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0033] In this document, the terms “containing,” “comprising,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0034] In this article, the term "H3.3K27M mutant diffuse endophytic pontine glioma" refers to a malignant tumor that occurs in the brainstem, particularly in the pons. This tumor primarily affects children and typically has a very poor prognosis. The H3.3K27M mutation occurs in the third subtype (H3.3) of histone H3, specifically at the 27th lysine residue (K). The normal lysine is mutated to methionine (M). This mutation affects the function of histone H3, leading to alterations in chromatin structure and gene expression. DIPGs primarily occur in the pons of the brainstem and control many essential physiological functions, such as respiration and heartbeat.

[0035] In this paper, the term "ABBV-075" refers to a small molecule inhibitor of BRD4, the chemical structure of which is described herein. ABBV-075 inhibits the ability of BRD4 to recognize histone acetylation by binding to the WPF domain of the BRD4 protein, thereby reducing the level of H3K27ac histone modification, inhibiting CREB5 expression, and subsequently killing brain tumor cells or promoting their differentiation.

[0036] In this article, the term "CREB5" refers to a transcription factor. CREB5 is highly expressed in diffuse endophytic pontine gliomas (DIPG) and is associated with tumor malignancy, stemness maintenance, and poor prognosis.

[0037] In this paper, the term "ID1" refers to a stemness-related gene involved in cell differentiation and tumor progression. ID1 is regulated by CREB5, and its expression level is associated with the tumor characteristics of DIPG.

[0038] In this paper, the term "BRD4" refers to a chromatin reader protein that recognizes acetylated histones (such as H3K27ac) and regulates gene transcription. BRD4 is enriched in the super-enhancer region of CREB5 and is associated with its activation.

[0039] In this article, the term "H3K27M" refers to the Histone H3 lysine 27-to-methionine mutation, commonly found in diffuse midline gliomas (DMGs), particularly DIPGs. This mutation leads to decreased genome-wide H3K27me3 levels and transcriptional abnormalities, which are associated with tumor invasiveness.

[0040] In this article, the term "DMG" refers to diffuse midline glioma, a WHO-classified malignant tumor of the central nervous system that occurs in midline structures such as the brainstem, thalamus, and spinal cord, and exhibits invasive behavior.

[0041] In this paper, the term "BRG1" refers to the catalytic subunit of the SWI / SNF chromatin remodeling complex, encoded by the SMARCA4 gene. BRG1 works synergistically with CREB5 to regulate the expression of tumor-related genes.

[0042] In this paper, the term "BRM014" refers to a small molecule inhibitor of BRG1 with the chemical structure described herein. BRM014 exhibits a synergistic effect when used in combination with ABBV-075, enhancing the killing effect on DIPG cells.

[0043] In this paper, the term "superenhancer" refers to a tightly clustered set of enhancer sequences that drive the high expression of key genes such as CREB5. Superenhancers are typically enriched with regulatory factors such as BRD4 and H3K27ac.

[0044] In this paper, the term "H3K27ac" refers to the acetylation of lysine 27 at position 27 of histone H3, an active histone marker associated with gene transcription activation.

[0045] In this article, the term "qPCR" refers to Quantitative Polymerase Chain Reaction, used to quantitatively detect gene expression levels.

[0046] In this article, the term "RNA-seq" refers to RNA sequencing, which is used for whole transcriptome analysis to detect gene expression profiles.

[0047] In this paper, the term "ChIP-seq" refers to chromatin immunoprecipitation sequencing, which is used to analyze the interaction between proteins (such as BRD4 and H3K27ac) and DNA.

[0048] In this paper, the term "Western Blot" refers to Western blotting, which is used to detect the expression and modification levels of a specific protein.

[0049] In this article, the term "PDX mouse" refers to a patient-derived xenograft model in which patient tumor cells are transplanted into immunodeficient mice to mimic tumor growth and drug response in vivo.

[0050] In this article, the term "IC50" refers to the half-maximal inhibitory concentration, which is the concentration at which a drug inhibits cell growth or activity by 50%, and is used to evaluate drug efficacy.

[0051] In this article, the term "JQ1" refers to a BRD4 small molecule inhibitor, commonly used as a positive control, whose mechanism of action is similar to that of ABBV-075.

[0052] In this article, the term "PPC cells" refers to a type of pontine precursor cells used as a normal cell control to assess the toxicity of the drug to normal tissues.

[0053] In this paper, the term "NCG mouse" refers to an immunodeficient mouse model (NOD / ShiLtJGpt-Prkdcem26Cd52II2rgem26Cd22 / Gpt) used for in vivo tumor experiments, such as DIPG PDX mice.

[0054] In this paper, the term "astrocytocyte" refers to a normal type of glial cell that participates in the supportive functions of the central nervous system. In this invention, "promoting the differentiation of brain tumor cells into astrocytes" refers to inducing brain tumor cells to differentiate into normal cell types, thereby reducing malignant characteristics.

[0055] This invention proposes a method for killing brain tumor cells in vitro, a method for targeting brain tumor cells in vitro, the use of reagents in the preparation of reagent kits, the use of reagents in the preparation of drugs, a pharmaceutical composition, a method for inhibiting CREB5 expression, a method for treating brain tumors, and a method for promoting the differentiation of brain tumor cells into astrocytes. These will be described in detail below.

[0056] A method for killing brain tumor cells in vitro

[0057] In a first aspect, the present invention provides a method for in vitro killing of brain tumor cells. According to an embodiment of the invention, the method includes: contacting isolated brain tumor cells with ABBV-075 to kill the brain tumor cells. The method according to the embodiment of the invention can improve the efficiency of in vitro killing of brain tumor cells.

[0058] According to an embodiment of the present invention, the structure of ABBV-075 is as follows:

[0059] According to embodiments of the present invention, the concentration of ABBV-075 is 1 nM to 1000 nM, for example, it can be 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, or a range between the two, such as 10 nM to 1000 nM or 100 nM to 1000 nM. Too low a concentration of ABBV-075 reduces the drug's tumor-killing effect, while too high a concentration causes toxic side effects on normal tissues. The method according to embodiments of the present invention can improve the efficiency of in vitro killing of brain tumor cells.

[0060] According to an embodiment of the present invention, the brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

[0061] According to embodiments of the present invention, the brain tumor cells include H3.3K27M mutant diffuse intrinsic pontine glioma cells. H3.3K27M mutant diffuse intrinsic pontine glioma (DIPG) is a malignant tumor occurring in the pons region of the brainstem in children, characterized by aggressive growth, difficulty in surgical resection, lack of effective treatment, and extremely poor prognosis. The H3.3K27M mutation is a common histone mutation in DIPG and is associated with poor prognosis. This mutation leads to a reduction in genome-wide H3K27me3 and transcriptional abnormalities, and is considered a major contributing factor to the pathogenesis of DIPG. Treatment for DIPG typically includes radiotherapy, but the median survival remains short, approximately 9 to 12 months, with a 2-year survival rate of approximately 10% to 25% and a 5-year survival rate of less than 1%. Patients with H3.3K27M mutant DIPG generally have a worse prognosis, and there are currently no particularly effective treatments for this type of tumor. According to an embodiment of the present invention, ABBV-075 is contacted with isolated H3.3K27M mutant diffuse endophytic pontine glioma cells to kill the H3.3K27M mutant diffuse endophytic pontine glioma cells.

[0062] According to an embodiment of the present invention, the method further includes contacting the BRG1 inhibitor with isolated brain tumor cells.

[0063] According to an embodiment of the present invention, the BRG1 inhibitor includes BRM014.

[0064] According to an embodiment of the present invention, the structure of BRM014 is as follows:

[0065] A method for targeting brain tumor cells in vitro

[0066] In a second aspect, the present invention provides a method for targeting brain tumor cells in vitro. According to an embodiment of the invention, the method includes: contacting ABBV-075 with a separated cell population, said cell population including brain tumor cells. The method according to the embodiment of the invention can specifically target brain tumor cells in vitro.

[0067] According to embodiments of the present invention, the concentration of ABBV-075 is 1 nM to 1000 nM, for example, it can be 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, or a range between the two, such as 10 nM to 1000 nM or 100 nM to 1000 nM. Too low a concentration of ABBV-075 reduces the drug's tumor-killing effect, while too high a concentration causes toxic side effects on normal tissues. The method according to embodiments of the present invention can specifically target brain tumor cells in vitro.

[0068] According to an embodiment of the present invention, the brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

[0069] According to an embodiment of the present invention, the brain tumor cells include H3.3K27M mutant diffuse endogenous pontine glioma cells.

[0070] According to an embodiment of the present invention, the cell population further includes normal brain cells.

[0071] According to an embodiment of the present invention, the normal brain cells include at least one of glial cells and neuronal cells.

[0072] Use of reagents in the preparation of reagent kits

[0073] In a third aspect, the invention provides for the use of the reagent in the preparation of a kit. According to embodiments of the invention, the reagent comprises ABBV-075, and the kit is used to inhibit the expression of CREB5 and / or ID1.

[0074] Uses of reagents in drug preparation

[0075] In a fourth aspect, the invention provides for the use of the reagent in the preparation of a medicament. According to embodiments of the invention, the reagent comprises ABBV-075, and the medicament is used to treat or prevent gliomas.

[0076] According to embodiments of the present invention, the reagent further comprises a BRG1 inhibitor. According to embodiments of the present invention, ABBV-075 and the BRG1 inhibitor work synergistically to improve the efficiency of killing gliomas.

[0077] According to an embodiment of the present invention, the BRG1 inhibitor includes BRM014. According to an embodiment of the present invention, ABBV-075 and BRM014 work synergistically to improve the efficiency of killing gliomas.

[0078] According to an embodiment of the present invention, the glioma occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

[0079] According to an embodiment of the present invention, the glioma includes an H3.3K27M mutant diffuse endophytic pontine glioma.

[0080] Pharmaceutical Composition

[0081] In a fifth aspect, the present invention provides a pharmaceutical composition for treating glioma. According to an embodiment of the invention, the pharmaceutical composition comprises ABBV-075. The pharmaceutical composition according to embodiments of the invention is capable of improving the efficiency of killing glioma cells in vitro and improving the specificity of targeting glioma cells.

[0082] According to an embodiment of the present invention, the pharmaceutical composition further comprises a BRG1 inhibitor. According to an embodiment of the present invention, ABBV-075 and the BRG1 inhibitor work synergistically to improve the efficiency of in vitro killing of brain tumor cells and enhance the specificity of targeting brain tumor cells.

[0083] According to an embodiment of the present invention, the BRG1 inhibitor includes BRM014. According to an embodiment of the present invention, ABBV-075 and BRM014 work synergistically to improve the efficiency of killing brain tumor cells in vitro and to enhance the specificity of targeting brain tumor cells.

[0084] According to embodiments of the present invention, the ABBV-075 and the BRG1 inhibitor are suitable for simultaneous or continuous administration.

[0085] According to an embodiment of the present invention, the glioma occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

[0086] According to an embodiment of the present invention, the glioma includes an H3.3K27M mutant diffuse endophytic pontine glioma.

[0087] Methods to inhibit CREB5 expression

[0088] In a sixth aspect, the present invention provides a method for inhibiting CREB5 expression. According to an embodiment of the invention, the method involves contacting ABBV-075 with isolated cells expressing CREB5 to inhibit the expression of said CREB5. The method according to an embodiment of the invention is capable of inhibiting CREB5 expression.

[0089] According to an embodiment of the present invention, the cells expressing CREB5 include brain tumor cells.

[0090] According to an embodiment of the present invention, the brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

[0091] According to an embodiment of the present invention, the brain tumor cells include H3.3K27M mutant diffuse endogenous pontine glioma cells.

[0092] According to an embodiment of the present invention, the inhibition of CREB5 expression is carried out by at least one of the following methods: the activity of the CREB5 superenhancer is inhibited; the level of H3K27ac histone modification is reduced.

[0093] Methods to promote the differentiation of brain tumor cells into astrocytes

[0094] In a seventh aspect, the present invention provides a method for promoting the differentiation of brain tumor cells into astrocytes. According to an embodiment of the invention, the method includes contacting isolated brain tumor cells with ABBV-075 to promote the differentiation of the brain tumor cells into astrocytes. The method according to an embodiment of the invention is capable of promoting the differentiation of brain tumor cells into astrocytes.

[0095] According to an embodiment of the present invention, the brain tumor cells include H3.3K27M mutant diffuse endogenous pontine glioma cells.

[0096] According to an embodiment of the present invention, the brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

[0097] Treatment methods for brain tumors

[0098] In an eighth aspect, the present invention provides a method for treating brain tumors. According to an embodiment of the invention, the method includes administering ABBV-075 to the subject. The method according to an embodiment of the invention is capable of effectively treating brain tumors.

[0099] According to an embodiment of the invention, the method further includes administering or exposing the subject to a BRG1 inhibitor of isolated brain tumor cells.

[0100] According to an embodiment of the present invention, the BRG1 inhibitor includes BRM014.

[0101] According to embodiments of the present invention, the ABBV-075 and the BRG1 inhibitor are suitable for simultaneous or continuous administration.

[0102] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0103] Example 1:

[0104] CREB5 is a malignant oncogenic factor specifically highly expressed in patients with diffuse endophytic pontine gliomas (DIPGs). Knockdown of CREB5 significantly inhibits the proliferation of DIPG cells, promotes their differentiation, and thus suppresses tumor growth in vivo. Furthermore, as an upstream regulator of the tumor stemness regulator ID1 in the H3.3K27M subtype, CREB5 can also collaborate with the chromatin remodeling complex SWI / SNF to regulate the expression of a series of genes associated with poor prognosis in cancer patients. Therefore, further targeting of CREB5 holds promise for providing better therapeutic outcomes for DIPG tumor patients.

[0105] This embodiment, through combined analysis of the transcriptional expression profile of CREB5 knockdown and the Connectivity Map (L1000), found that the small molecule inhibitor of BRD4, JQ1 (HY-13030, MCE), has a similar effect to CREB5 knockdown.

[0106] First, this embodiment analyzed ChIP-seq data (NCBI GEO database, https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE78801) of the H3.3K27M and H3.1K27M isotypes, and found that the chromatin reader proteins BRD2 and BRD4 were heavily enriched in the super-enhancer region of CREB5, and histone active modification markers H3K27ac and oncohistone H3.3K27M were present. However, these phenomena were not observed in DIPG cells of the H3.1K27M isotype, which is consistent with the fact that the expression level of CREB5 in DIPG cells of the H3.1K27M isotype is lower than that in DIPG cells of the H3.3K27M isotype.

[0107] Secondly, the RNA-seq data of DIPG cells treated with the common BRD4 inhibitor JQ1 were analyzed, and the results are shown in Figure 1. In this example, it was found that the expression level of CREB5 in H3.3K27M subtype DIPG cells decreased with the extension of JQ1 treatment time, which further verified that BRD4 inhibitors and CREB5 knockdown have similar expression profiles.

[0108] Next, this embodiment used qPCR to detect the expression of CREB5 and some stem cell-related genes (OLIG2, SOX2, PDGFRA, PROM1) after JQ1 treatment. The results are shown in Figure 2. The results indicate that JQ1 treatment can indeed significantly downregulate the expression of CREB5 and some other stem cell genes. Therefore, these experimental data show that small molecule inhibitors of BRD4 can significantly inhibit the expression of CREB5, which is more important for further screening of more effective small molecule inhibitors of BRD4 for targeting the CREB5 transcription factor.

[0109] Example 2:

[0110] To identify potential small molecule inhibitors of BRD4 targeting CREB5 superenhancer activation, this embodiment screened the MCE active compound library. The screening strategy was as follows: 1. Small molecule inhibitors targeting BRD4 activity were identified from the MCE active compound library; 2. The inhibitory level of BRD4 small molecule inhibitors on CREB5 was evaluated in DIPG cells using qPCR experiments; 3. Safe and reliable BRD4 small molecule inhibitors were screened by comparing the cell viability of small molecule inhibitors on normal pontine progenitor cells (PPC cells) and DIPG cells.

[0111] This embodiment first identifies 18 BRD4 small molecule inhibitors (HY-13960, HY-111422, HY-112610, HY-13235, HY-100015, HY-13959, HY-112149, HY-101146, HY-111102, HY-112718, HY-16586, HY-16954, HY-50698, HY-103633, HY-101838, HY-125232, HY-126428, HY-100653A, MCE) from the MCE active compound library. qPCR experiments revealed that five small molecule inhibitors (HY-13960, HY-111422, HY-112610, HY-13235, HY-100015, MCE) significantly and effectively inhibited CREB5 expression, as shown in Figure 3. Finally, the cytotoxic effects of these five small molecule inhibitors on DIPG and PPC cells were examined, as shown in Figure 4. Only the fifth small molecule inhibitor (HY-100015, also known as ABBV-075) significantly inhibited DIPG cell growth and showed no significant toxic side effects on PPC cells.

[0112] Example 3:

[0113] To enhance the clinical application potential of ABBV-075 in the treatment of DIPG, this embodiment aims to further clarify the mechanism of action of ABBV-075 in inhibiting CREB5 expression by suppressing the activity of the CREB5 super-enhancer. This embodiment utilizes molecular docking experiments, and the results, shown in Figure 5, reveal that ABBV-075 has a similar ability to bind to the WPF domain of the BRD4 protein as JQ1, predicting that ABBV-075 can inhibit the ability of BRD4 to recognize histone acetylation.

[0114] Therefore, this embodiment uses Western blotting to show that treatment with different times and concentrations of ABBV-075 significantly downregulates the H3K27ac histone modification level, as shown in Figures 6 and 7. In this process, this embodiment also found that ABBV-075 downregulates not only CREB5 protein levels but also OLIG2 and ID1 protein levels, inducing neurite-like differentiation phenotypes in DIPG cells, as shown in Figure 8.

[0115] These results corroborate that ABBV-075 can serve as an effective small molecule inhibitor of BRD4, thereby inhibiting CREB5 expression by reducing H3K27ac levels and further promoting DIPG cell differentiation.

[0116] Meanwhile, this embodiment also constructed a dual-fluorescent reporter system for CREB5 super-enhancer and ordinary enhancer along with their promoters to evaluate the specific molecular mechanism of ABBV-075 on CREB5. Consistent with the prevalent presence of super-enhancers and highly active transcription of CREB5 in DIPG cells, the super-enhancer of CREB5 significantly promotes CREB5 promoter activity compared to the ordinary enhancer, as shown in Figure 9. After treatment with ABBV-075, the promoting effect of the CREB5 super-enhancer on its promoter activity was significantly reduced, indicating that ABBV-075 can inhibit CREB5 expression by inhibiting the activity of the CREB5 super-enhancer and promoter, as shown in Figure 10. Using concentration-inhibition curves, this embodiment found that ABBV-075 can significantly inhibit the activity of the CREB5 enhancer at the 20 nM level, as shown in Figure 11, further emphasizing its higher inhibitory concentration and fewer toxic side effects compared to other BRD4 small molecule inhibitors. ABBV-075 is a very potent BRD4 small molecule inhibitor for the treatment of DIPG.

[0117] Example 4:

[0118] This embodiment first evaluated the therapeutic effect of ABBV-075 on DIPG cells in vitro. Using JQ1 as a positive control, growth curves and spheroidization assays were used to detect the killing effect of ABBV-075 on different DIPG cell types. The results showed that ABBV-075 had less toxicity to normal PPC cells compared to JQ1, while exhibiting more effective killing of DIPG glioma cells and a lower IC50 value, as shown in Figure 12. This indicates that ABBV-075 has a highly selective killing ability against DIPG cells compared to JQ1, and lower toxicity to PPC cells, which are similar in state to DIPG cells.

[0119] Therefore, this embodiment further evaluated the anticancer effect of ABBV-075 in vivo on PDX mice injected with DIPG in situ TT150630 cells. In this embodiment, TT150630 cells derived from DIPG patients were first injected into immunodeficient mice (NCG mice) (5 control mice and 5 treatment mice, for a total of 10 mice; the mice were derived from Nanjing Jicui Pharmaceutical Co., Ltd., NOD / ShiLtJGpt-Prkdcem26Cd52II2rgem26Cd22 / Gpt (NCG mice)) in the pons to induce tumor formation for 14 days. Then, the mice were administered the drug orally via gavage every three days at a concentration of 1 mg / kg for a total of 8 times. Tumor cell growth was monitored weekly using in vivo fluorescence imaging. As shown in Figure 13, the results of biofluorescence imaging showed that, compared to the saline control group, the growth of DIPG tumor-bearing mice in the ABBV-075 treatment group was significantly inhibited, and their survival time was significantly longer. These results indicate that ABBV-075 can significantly inhibit the growth of DIPG tumors in vivo.

[0120] Considering that CREB5 in DIPG cells can synergistically exert a pro-cancer effect with the catalytic subunit BRG1 of the SWI / SNF chromatin remodeling complex, and that existing technologies have disclosed the anti-tumor effects of BRG1 inhibitors on DIPG cells, this embodiment further evaluated the therapeutic effects of ABBV-075 and the BRG1 small molecule inhibitor (BRM014) on DIPG cells. Using a combination index model and a Bliss-independent model, this embodiment evaluated the combined therapeutic effect of ABBV-075 and BRM014 on DIPG17 and TT150714 cells. At lower concentrations, the combination therapy showed a highly significant inhibitory effect in both DIPG cell types. By calculating the therapeutic index for each combination using Compusyn software, this embodiment identified synergistic therapeutic regions within the combination therapy matrix. Further calculation of the combined synergistic therapeutic score of the two small molecule inhibitors revealed a highly significant synergistic therapeutic effect in both DIPG17 (Bliss: 12.04) and TT150714 (Bliss: 16.01) cells, as shown in Figure 14. Therefore, based on all the above results, this embodiment demonstrates that ABBV-075 exerts its anti-tumor effect by inhibiting CREB5 super-enhancer activity, and exhibits low toxicity to normal neural stem cells. Furthermore, the combination of ABBV-075 and a BRG1 small molecule inhibitor showed a significant combined therapeutic effect on H3.3K27M DIPG cells.

[0121] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0122] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for killing brain tumor cells in vitro, characterized in that, include: ABBV-075 was applied to isolated brain tumor cells to kill them.

2. A method for targeting brain tumor cells in vitro, characterized in that, include: ABBV-075 was contacted with a separated cell population, including brain tumor cells.

3. The method according to claim 1 or 2, characterized in that, include: The concentration of ABBV-075 is 1 nM to 1000 nM.

4. The method according to claim 1 or 2, characterized in that, include: The brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

5. The method according to claim 1 or 2, characterized in that, include: The brain tumor cells include H3.3K27M mutant diffuse endogenous pontine glioma cells.

6. The method according to claim 2, characterized in that, The cell population further includes normal brain cells.

7. The method according to claim 6, characterized in that, The normal brain cells include at least one of glial cells and neurons.

8. The method according to claim 1, characterized in that, Further, it includes contact treatment of BRG1 inhibitors with isolated brain tumor cells.

9. The method according to claim 8, characterized in that, The BRG1 inhibitor includes BRM014.

10. Use of the reagent in the preparation of the kit, the reagent comprising ABBV-075, the kit being used to inhibit the expression of CREB5 and / or ID1.

11. Use of the reagent in the preparation of a medicament, said reagent comprising ABBV-075, said medicament for the treatment and / or prevention of glioma.

12. The use according to claim 11, characterized in that, The reagent further includes a BRG1 inhibitor.

13. The use according to claim 12, characterized in that, The BRG1 inhibitor includes BRM014.

14. The use according to claim 11, characterized in that, The glioma occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

15. The use according to claim 11, characterized in that, The gliomas include H3.3K27M mutant diffuse endophytic pontine gliomas.

16. A pharmaceutical composition for treating or preventing glioma, characterized in that, include: ABBV-075.

17. The pharmaceutical composition according to claim 16, characterized in that, Further includes: BRG1 inhibitors.

18. The pharmaceutical composition according to claim 17, characterized in that, The BRG1 inhibitor includes BRM014.

19. The pharmaceutical composition according to claim 16 or 17, characterized in that, The ABBV-075 and the BRG1 inhibitor are suitable for simultaneous or continuous administration.

20. The pharmaceutical composition according to claim 16, characterized in that, The glioma occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

21. The pharmaceutical composition according to claim 16, characterized in that, The gliomas include H3.3K27M mutant diffuse endophytic pontine gliomas.

22. A method for inhibiting CREB5 expression, characterized in that, ABBV-075 was contacted with isolated cells expressing CREB5 in order to inhibit the expression of CREB5.

23. The method according to claim 22, characterized in that, The cells expressing CREB5 include brain tumor cells.

24. The method according to claim 23, characterized in that, The brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

25. The method according to claim 23, characterized in that, The brain tumor cells include H3.3K27M mutant diffuse endogenous pontine glioma cells.

26. The method according to claim 22, characterized in that, Suppressing the expression of CREB5 is achieved through at least one of the following methods: The activity of the CREB5 super enhancer was suppressed; The level of H3K27ac histone modification was reduced.

27. A method for promoting the differentiation of brain tumor cells into astrocytes, characterized in that, include: ABBV-075 was applied to isolated brain tumor cells to promote their differentiation into astrocytes.

28. The method according to claim 27, characterized in that, The brain tumor cells include H3.3K27M mutant diffuse endogenous pontine glioma cells.

29. The method according to claim 27, characterized in that, The brain tumor occurs in at least one of the spinal cord, brainstem, pineal region, and thalamus.

30. A method for treating brain tumors, characterized in that, This includes administering ABBV-075 to the subjects.

31. The method according to claim 30, characterized in that, Further, it includes administering or exposing the subject to a BRG1 inhibitor.

32. The method according to claim 31, characterized in that, The BRG1 inhibitor includes BRM014.

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