Application of MYC inhibitors in the preparation of BET inhibitor sensitizing drugs

Abstract

This invention relates to the field of biopharmaceuticals and discloses the use of MYC inhibitors in the preparation of drugs that enhance BET inhibitor sensitization. To identify synergistic agents with BET inhibitors, the present invention systematically screened for core transcription factors regulating super-enhancers (SEs) and discovered that MYC is a core master transcription factor in the tumor SE regulatory network. Furthermore, the present invention experimentally demonstrated that inducing degradation of endogenous MYC protein, inducing expression of the dominant-negative MYC inhibitor peptide Omomyc, or treating with a MYC small molecule inhibitor significantly enhances the tumor suppressor effect of BET inhibitors and reduces their effective concentration. Therefore, MYC inhibitors can be used as BET inhibitor sensitizers and have promising application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a MYC inhibitor in the preparation of a BET inhibitor sensitizing drug. Background Art

[0002] Among eukaryotic transcriptionally active cis-regulatory elements, super-enhancers (SEs), whose activity is significantly higher than that of standard enhancers, have been found to be key regulators of cell fate-determining genes and control the transcriptional output of numerous disease-associated genes. Compared to standard enhancers, which range from hundreds to thousands of bases in size, SEs typically span a vast region of 10 to 20 kb, may contain multiple enhancer clusters, and are highly enriched for transcription factors and chromatin regulators. Epigenomic studies in colorectal cancer (CRC), gastric cancer, nasopharyngeal carcinoma, and various other epithelial tumors have clearly implicated aberrant SE activity as a key driver of carcinogenesis. Studies have shown that genes controlled by SEs are more sensitive to changes in external environmental signals than ordinary genes, making selective targeting of SEs a promising therapeutic option. Indeed, small molecule drugs (BETis) that inhibit BET (Bromodomain and extraterminal) domain proteins have demonstrated promising tumor suppressor effects in preclinical studies, and treatment with these drugs selectively downregulates the expression of SE-regulated oncogenes. BET family proteins, including BRD2, BRD3, BRD4, and BRDT, act as epigenetic readers and are considered key therapeutic targets due to their ability to recognize and bind acetylated histones (such as H3K27ac) and their important role in SE activity. BET inhibitors (BETi) developed based on this compete with BET protein domains, blocking their ability to recognize H3K27ac and transcription factors, thereby inhibiting tumor progression.

[0003] Despite promising results in numerous preclinical models and ongoing clinical trials for solid and hematologic cancers, BETi (beta-lactamase inhibitors) have yet to receive approval due to concerns about high-dose toxicity and drug resistance. For example, GSK3358699 was eliminated due to serious adverse reactions, primarily headaches, occurring at doses below the intended therapeutic dose. OTX015, used in Phase I clinical trials for the treatment of acute myeloid leukemia and various solid tumors, was largely associated with anemia, neutropenia, and gastrointestinal symptoms such as nausea and diarrhea. iBET762, used in preclinical trials for the treatment of various tumors, including neuroblastoma and pancreatic cancer, exhibited similar toxicities to those of OTX015 in clinical trials, suggesting that these toxicities may be common with BETi. Current research to optimize these issues focuses on drug combinations, aiming to reduce BETi doses through synergistic combinations, thereby minimizing toxicities and achieving better therapeutic outcomes. Therefore, identifying therapeutic strategies for synergistic BETi is crucial. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the use of MYC inhibitors in the preparation of BET inhibitor sensitization drugs, aiming to solve the problem of toxic side effects of high doses of BET inhibitors during clinical application.

[0005] The technical solutions of the present invention are as follows:

[0006] In a first aspect, a method is provided for using a MYC inhibitor in the preparation of a BET inhibitor sensitizing drug.

[0007] In a preferred technical solution, the sensitizing drug is used to enhance the tumor therapeutic effect of the BET inhibitor.

[0008] In a further preferred technical solution, the tumor is colorectal cancer.

[0009] In a preferred technical solution, the BET inhibitor exerts its tumor therapeutic effect by targeting and binding to BRD4.

[0010] In a preferred technical solution, the BET inhibitor is selected from one or more of the following structures:

[0011] .

[0012] In a preferred technical solution, the MYC inhibitor is selected from one or more of the following structures:

[0013] .

[0014] In a preferred technical solution, the MYC inhibitor is a polypeptide having an amino acid sequence as shown in SEQ ID NO.1.

[0015] In a second aspect, an anti-tumor composition is provided, wherein the anti-tumor composition comprises a BET inhibitor and a sensitizing drug, wherein the sensitizing drug comprises a MYC inhibitor.

[0016] In a preferred technical solution, the anti-tumor composition further comprises a pharmaceutically acceptable carrier.

[0017] Beneficial Effects: This invention provides the use of MYC inhibitors in the preparation of BET inhibitor sensitizers, proposing for the first time that simultaneously targeting MYC and BRD4 can synergistically inhibit oncogenic transcription and thereby block tumor growth. Experiments have shown that inducing degradation of endogenous MYC protein, inducing expression of the dominant-negative inhibitory peptide Omomyc of MYC, or treating with MYC small molecule inhibitors can significantly enhance the tumor suppressor effect of BET inhibitors and reduce their effective concentrations. Therefore, MYC inhibitors can be used as BET inhibitor sensitizers and have promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the identification of MYC as a core transcription factor regulating SE activity; A is the transcription factor motif enrichment spectrum in the SE region across cancer types, based on SE motif enrichment analysis of 14 tumor cell lines; B is the occupancy preference of MYC in cis-regulatory elements, which shows the percentage of cis-elements occupied by MYC in cancer cell lines from different sources (there is at least one MYC ChIP-seq peak in the element region obtained from the same cell line), with each point representing a cell line; C is the co-localization feature of MYC and SE activity markers, which visualizes the ChIP-seq signal intensities of H3K27ac, MYC, MED1, and BRD4 after sorting enhancers based on H3K27ac signal intensity in K562 cells.

[0019] Figure 2 This is an analysis result diagram showing that MYC is preferentially enriched in tumor type-specific SEs; A is the distribution of various factors on SEs occupied by MYC across cell lines; B is the visualization of ChIP-seq through IGV, showing the enrichment of MYC on its own gene locus SEs in different tumor cell types.

[0020] Figure 3 Figure 1 is a diagram showing the construction and validation of an endogenous MYC inducible degradation system; A is a schematic diagram of the MYC-AID protein degradation construction; B is the Western blot analysis of protein cell lysates obtained by treating the MYC-AID cell line established in HT29 or LoVo with 500 μM IAA for designated time points or 3 hours of IAA treatment followed by PBS washing; HA antibody was used to detect endogenous MYC protein fused to the HA tag, which produced results consistent with those of the MYC antibody; GAPDH was used as a control.

[0021] Figure 4 This is the GSEA result of HT29-MYC-AID cells targeting only SE genes.

[0022] Figure 5 The following is a diagram showing the verification results of the coordinated occupancy of MYC and BRD4 in the SE region; A is a schematic diagram of the functional interaction mechanism between MYC and BRD4; B is the result of co-immunoprecipitation (Co-IP) experiments in HT29-MYC-AID and LoVo-MYC-AID cells, which serves as a verification of the endogenous interaction between MYC and BRD4; C is a scatter plot of the correlation between MYC and BRD4 enrichment on SEs, reflecting the strong correlation between MYC and BRD4 occupancy in the SE region; D is the binding ratio of MYC and BRD4 in the SE region.

[0023] Figure 6 This is a verification result diagram of MYC regulating the enrichment of BRD4 in the SE region; among them, A is the BRD4 ChIP-seq signal in the SE region occupied or not by MYC; B is the ChIP-seq signal of BRD4 in the SE region after MYC degradation was induced by IAA in HT29-MYC-AID and LoVo-MYC-AID cells; C is the ChIP-seq signal intensity heat map of the indicator factors within a 10kb window centered on the enhancer in HT29-MYC-AID and LoVo-MYC-AID cells.

[0024] Figure 7 These are the results of the in vitro proliferation inhibition experiment, clone formation, and sphere formation experiment of the MYC-AID cell line in Example 3-5; wherein A is the result of the in vitro proliferation inhibition experiment; B is the result of the clone formation experiment; and C is the result of the sphere formation experiment.

[0025] Figure 8 These are the results of the detection of the TetOn-Omomyc inducible expression system cell line in Example 2 and the in vitro proliferation experiment results of the TetOn-Omomyc inducible expression system cell line in Examples 3-5; wherein, A is the protein blot analysis result of the TetOn-Omomyc inducible expression system cell line; B is the result of the in vitro proliferation inhibition experiment.

[0026] Figure 9 These are the results of the in vitro clone formation and sphere formation experiments of the TetOn-Omomyc inducible expression system cell line in Example 4-5; wherein A is the result of the clone formation experiment; and B is the result of the sphere formation experiment.

[0027] Figure 10 3 is a graph showing the results of an in vitro proliferation experiment of two inhibitors in a human colorectal cancer cell line in Example 3; wherein A is a partial specific result of the synergy of the two inhibitors; and B is a statistical result of the average synergy score of the two inhibitors. DETAILED DESCRIPTION

[0028] The present invention provides the use of a MYC inhibitor in the preparation of a BET inhibitor sensitizing drug. To make the objectives, technical solutions and effects of the present invention clearer and more specific, the present invention is described in further detail below.

[0029] BETi, which targets and inhibits BET family proteins, has demonstrated promising anti-cancer effects in preclinical studies. Multiple clinical trials are underway for both solid and hematologic cancers, but they remain unapproved due to concerns about high-dose toxicity and drug resistance. Therefore, identifying synergistic drugs to reduce BETi dosage and thus minimize toxicity is crucial.

[0030] To screen for synergistic drugs of BETi, the research scheme of the present invention is as follows:

[0031] 1. Identification of MYC as a master transcription factor regulating SE activity

[0032] To systematically screen for core transcription factors that regulate SE, this paper integrated SE region motif enrichment analysis of 14 cell lines (covering five hematological tumors and six solid cancers) and found that MYC was significantly enriched on SE in all cell lines ( Figure 1 Middle A), MYC occupancy maps at H3K27ac-defined SEs and typical enhancer (TE) regions were constructed by chromatin immunoprecipitation high-throughput sequencing (ChIP-seq). The results showed that the median proportion of MYC-bound SE regions reached 85% (interquartile range: 56%-100%), and this phenomenon was independent of cell lineage origin ( Figure 1 Middle B), suggesting that MYC may serve as the core master transcription factor of the tumor SE regulatory network.

[0033] To analyze the correlation between MYC and SE activity, the present invention first sorted genome-wide enhancers in K562 cells based on H3K27ac signal intensity and visualized the enrichment signals of MYC and SE regulatory proteins BRD4 and MED1 corresponding to each enhancer. The enrichment of MYC in enhancers was highly consistent with the enrichment of H3K27ac, BRD4, and MED1 related to enhancer activity, suggesting that MYC enrichment may be another biomarker of active SE ( Figure 1 Middle C).

[0034] 2. MYC selectively occupies tumor-type-specific SEs

[0035] One of the core characteristics of SE is its tissue context specificity. This dynamic regulatory property makes it a key condition for precisely controlling transcriptional output under specific conditions. ChIP-seq analysis of different types of tumor cell lines showed that MYC selectively occupies tumor type-specific SE ( Figure 2 Middle A), MYC simultaneously binds to the SE region of its own locus in a tumor-specific manner ( Figure 2 Middle B), indicating the existence of a positive feedback loop for MYC self-regulation.

[0036] 3. Inducing acute degradation of MYC protein in CRC cells selectively inhibits SE-driven transcription

[0037] To further explore the direct regulatory effect of MYC on SE activity, the present invention uses CRISPR / Cas9-mediated homology-directed repair technology to construct an auxin-inducible degron (AID) system targeting endogenous MYC protein in CRC cell lines HT29 and LoVo ( Figure 3 Western blot analysis (WB) verified the induction and degradation efficiency of MYC. The expression of endogenous MYC was almost completely degraded after 0.5 hours of auxin (IAA) induction, and the expression returned to the baseline level after 48 hours of IAA withdrawal ( Figure 3 Middle B), demonstrating that this system can achieve time-dependent manipulation of MYC activity.

[0038] By using the auxin-induced degradation system (MYC-AID) targeting the MYC protein, the present invention first classified MYC-regulated genes based on the differences in MYC binding strength between enhancers and promoters, and then performed gene set enrichment analysis (GSEA) on different gene sets based on the binding tendency of MYC at cis-acting elements. The results showed that compared with other MYC-regulated genes, the expression levels of SE-controlled genes with higher MYC binding strength were more sensitive to acute MYC degradation (Table 1), and their transcription levels recovered more significantly after MYC recovery ( Figure 4 These results demonstrate that MYC specifically regulates tumor cell transcriptional output through SE.

[0039] Table 1 GSEA results of four MYC-regulated gene sets classified by MYC binding preference

[0040]

[0041] 4. MYC and BRD4 jointly occupy SE active elements in tumor cells

[0042] BRD4 is one of the key SE regulatory factors known, and the above studies reveal the importance of MYC in regulating SE activity, suggesting the potential synergistic effect of the two in SE regulation, that is, the interdependence of their SE regulatory functions ( Figure 5 In Figure A, the present invention confirmed the physical interaction between MYC and BRD4 proteins by protein co-immunoprecipitation (Co-IP) ( Figure 5 Secondly, correlation analysis of the enrichment levels of MYC and BRD4 on SE revealed a strong positive correlation between the two ( Figure 5 In addition, genome-wide chromatin occupancy profiling showed that the binding locations of BRD4 and MYC in cancer cells from different tissues were highly consistent across the genome ( Figure 5 Middle D).

[0043] 5. BRD4 enrichment in SE regions is partially dependent on MYC

[0044] In CRC cells, BRD4 is significantly more enriched in SE elements occupied by MYC than in SE elements not occupied by MYC ( Figure 6 Middle A), and acute degradation of MYC leads to a decrease in the binding ability of BRD4 in the SE region ( Figure 6 (B, C) suggest that MYC and BRD4 form functional synergy through physical interaction and SE co-localization, and MYC is one of the conditions for BRD4 to be recruited to SE, providing a theoretical basis for a synergistic combination therapy strategy that simultaneously targets both.

[0045] Based on the above research, embodiments of the present invention provide the use of MYC inhibitors in the preparation of BET inhibitor sensitization drugs.

[0046] Specifically, MYC, one of the most representative genes controlled by SEs across multiple cancer types, has long been considered to function downstream of the SE regulatory machinery (e.g., BRD proteins), primarily acting as an effector that amplifies the SE transcriptional cascade. Furthermore, as a master transcription factor, MYC is crucial for maintaining normal cell proliferation. When overactivated, it can lead to cancerous transformation. In CRC, MYC overactivation may be due to APC gene mutations or MYC gene copy number amplification. This breakthrough reveals that MYC can enrich and bind to tumor-type-specific SEs and actively regulate SE activity, as well as a functional interdependence between MYC and BRD4 in controlling SE-driven oncogenic transcription. Based on this, it is proposed that simultaneous targeting of MYC and BRD4 can synergistically inhibit oncogenic transcription and thereby block tumor growth. Using colorectal cancer (CRC) as a model, this study intervened in MYC activity in CRC cells through three different approaches: inducing endogenous MYC protein degradation, inducing expression of the dominant-negative MYC inhibitor Omomyc, or treating with a MYC small molecule inhibitor. The results consistently demonstrated that decreased MYC activity effectively sensitized CRC cells to BET inhibitors. Therefore, MYC inhibitors can be used to prepare BET inhibitor sensitizing drugs.

[0047] Currently, targeted intervention approaches for MYC fall into two main categories. One involves the use of small molecule MYC inhibitors (MYCi), which disrupt the binding of the MYC / MAX complex and thereby exert their effects. For example, MYCi975 binds to the carboxy-terminal bHLHZip domain of MYC and disrupts the formation of the MYC / MAX complex. The other involves the 91-amino acid small peptide Omomyc (SEQ ID NO. 1: MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA, PDB ID pdb_00005i50). As a dominant-negative MYC mutant, Omomyc contains the carboxy-terminal bHLHZip domain of MYC, with four point mutations in the leucine zipper: 63T, 70I, 76Q, and 77N. These mutations alter the dimerization specificity of Omomyc, allowing it to bind to MAX as a homodimer or heterodimer. This binding enables Omomyc to compete with the MYC / MAX complex for recognition and binding to the E-box DNA sequence. Through this intermolecular competition, Omomyc significantly reduces the binding affinity of the MYC / MAX complex to the promoter region of its target gene, thereby inhibiting MYC-mediated transcriptional activation. At the same time, Omomyc has been shown to exhibit anti-cancer activity in various preclinical models. OMO-103, currently developed based on Omomyc, has fully passed Phase I clinical trials and has potential efficacy and acceptable toxicity.

[0048] The present invention will be further described below with reference to specific examples.

[0049] Example 1 Construction of MYC protein induced degradation system (AID) in CRC cells

[0050] 1. Experimental Materials

[0051] Human colorectal cancer cell lines HT29 (MYC gene amplification, APC gene mutation), LoVo (MYC gene non-amplification, APC gene mutation), RPMI1640 medium, transfection reagent, plasmid, serum, and double-antibody.

[0052] 2. Experimental methods

[0053] (1) Construction of a stable line overexpressing auxin receptor F-box protein (AtAFB)

[0054] ① Co-transfect the plasmids P-AtAFB2-mCherry-weak NLS (Addgene, Catalog No.: 129717) and pCas9-sgAAVS1-1 (Addgene, Catalog No.: 129726) into HT29 and LoVo cells, respectively.

[0055] ② Antibiotics were used to screen positive cells, and the transfection efficiency was detected before amplification and culture. Single clones were selected to establish stable strains, and HT29 and LoVo cells stably expressing AtAFB2 were obtained.

[0056] (2) The degradation subtag minilAA7 is integrated into the target gene MYC

[0057] ① Design sgRNA at the stop codon position of the target gene.

[0058] ②Clone the sgRNA into the pre-enzyme-cut lentiCRISPR v2 vector (Addgene, Cat. No. 52961) to obtain the sgRNA recombinant plasmid.

[0059] ③Construct a homologous repair plasmid containing the left homologous arm-miniIAA7 and the right homologous arm-Blastidin resistance gene.

[0060] ④ Co-transfect the sgRNA recombinant plasmid and homologous repair plasmid into HT29 and LoVo cells that have stably expressed AtAFB2.

[0061] ⑤ After antibiotic screening and transfection efficiency detection, the cells were expanded and cultured, and monoclonal screening was performed to obtain HT29 cells with a MYC protein-induced degradation system (HT29-MYC-AID) and LoVo cells with a MYC protein-induced degradation system (LoVo-MYC-AID).

[0062] ⑥ The genomic DNA of HT29-MYC-AID and LoVo-MYC-AID was extracted respectively, and the correct integration of the miniIAA7 sequence was verified by PCR, and its induced degradation efficiency was verified by Western Blot detection.

[0063] 3. Experimental results

[0064] Test results such as Figure 3 As shown in B, WB results show that a MYC protein-induced degradation system was successfully constructed in MYC gene-amplified and non-amplified colorectal cancer cell lines, and this system can achieve time-dependent control of MYC activity.

[0065] Example 2 Construction of TetOn-Omomyc Inducible Expression System in CRC Cells

[0066] 1. Experimental Materials

[0067] Human colorectal cancer cell lines HT29 (MYC gene amplification, APC gene mutation), RKO (MYC gene amplification, APC gene wild-type), HCT15 (MYC gene non-amplification, APC gene mutation), CACO2 (MYC gene non-amplification, APC gene mutation), human renal epithelial cell line Lentix-293T (Clonetech, cat. no. 632180), DMEM and RPMI1640 culture media, PEI transfection reagent, plasmids, serum, and double-antibody.

[0068] 2. Experimental methods

[0069] (1) Virus packaging

[0070] ① One day in advance, seed Lentix-293T cells into a 6-well plate and culture with DMEM complete medium. The cells should reach 70%-80% confluency within 1 day.

[0071] ② 30 minutes before transfection, replace the DMEM complete medium in the plate with DMEM medium without antibiotics and serum.

[0072] ③ Add 200 μL of antibiotic- and serum-free DMEM medium, PEI transfection reagent, packaging plasmids (0.7 μg VSVG (Addgene, Catalog No.: 8454), 2.1 μg PAX2 (Addgene, Catalog No.: 12260)), and target plasmid (3.2 μg pLVX-TetOne-Omomyc (Clontech, Catalog No.: 631847)) to a 1.5 mL EP tube. Gently pipette to mix, and let stand at room temperature for 20 min to obtain a mixed solution.

[0073] ③ Gently drop the above mixture into the culture medium of Lentix-293T cells and place it in a cell culture incubator for 6-8 hours. Replace the culture medium with fresh complete medium and collect the cell supernatant 48 hours and 72 hours after the medium replacement. Combine and use to obtain the viral supernatant.

[0074] (2) Viral infection

[0075] ① One day in advance, seed the target cells to be infected into a 6-well plate and perform viral infection after the cell density reaches 40-50%.

[0076] ② After filtering the viral supernatant with a 0.45μm filter membrane, mix the viral supernatant filtrate with fresh culture medium at a volume ratio of 3:1 and add 5μg / mL polybrene. Then add it to a 6-well plate and place it in the incubator for 10-12 hours. Replace the culture medium with fresh complete culture medium.

[0077] ③ 48 hours after changing the medium, use corresponding antibiotics for screening and perform Western Blot detection.

[0078] 3. Experimental results

[0079] Test results such as Figure 8 As shown in A, WB results show that the TetOn-Omomyc inducible expression system was successfully constructed in MYC-amplified and non-amplified colorectal cancer cell lines. The system can express the MYC dominant-negative inhibitory peptide Omomyc with a ubiquitin protein (HA) tag. The amino acid sequence of Omomyc is shown in SEQ ID NO.1.

[0080] Example 3 CRC cell proliferation inhibition experiment

[0081] 1. Experimental Materials

[0082] Human colorectal cancer cell lines HT29 (MYC gene amplification, APC gene mutation), RKO (MYC gene amplification, APC gene wild type), HCT15 (MYC gene non-amplification, APC gene mutation), CACO2 (MYC gene non-amplification, APC gene mutation), the MYC gene successfully constructed in Example 1 above MYC-AID cell lines (HT29-MYC-AID, LOVO-MYC-AID) and the TetOn-Omomyc inducible expression system cell lines successfully constructed in Example 2 (RKO-TetOn-Omomyc, CACO2-TetOn-Omomyc, HT29-TetOn-Omomyc, HCT15-TetOn-Omomyc), RPMI1640 medium, doxycycline (Dox), IAA, BETi (JQ1, ARV-771, OTX015, iBET762), MYCi (MYCi975, KJ-Pyr-9, MYCMI-6).

[0083] 2. Experimental methods

[0084] ① Take the above cells in the logarithmic growth phase and seed them at a certain density in a 96-well plate. The in vitro proliferation experiment of the two inhibitors in the human colorectal cancer cell line can then proceed to step ②. The in vitro proliferation experiment of the TetOn-Omomyc inducible expression cell line and the MYC-AID cell line should be divided into two groups respectively. Set up five concentration gradients in each group, and each concentration gradient has six replicates. Add 100 μL of culture medium to each well. Group 1 is immediately induced by adding doxycycline or IAA (add 1 μg / mL doxycycline to the TetOn-Omomyc inducible expression cell line and 500 μM IAA to the MYC-AID cell line) and culture for 24 hours.

[0085] ② After 24 hours, discard the old culture medium and add culture medium containing different drug concentrations. The dosage concentration settings for the in vitro proliferation experiment of two inhibitors in human colorectal cancer cell lines are shown in Figure 10 In the in vitro proliferation experiments of TetOn-Omomyc-inducible expression cell lines and MYC-AID cell lines, BETi was added alone to group 2, and doxycycline or IAA was added to group 1 in addition to BETi (1 μg / mL doxycycline was added to the TetOn-Omomyc-inducible expression cell line, and 500 μM IAA was added to the MYC-AID cell line). The drug concentrations are shown in Table 1. Figure 7 Middle A, Figure 8 In B, the unit of drug concentration is μM.

[0086] ③ In vitro proliferation assay of two inhibitors in human colorectal cancer cell lines: After 48 hours of culture, the cells were replaced with RPMI1640 medium containing CCK-8 detection reagent (CCK-8: RPMI1640 = 1:10), and incubated in a 37°C incubator for 1 hour and 20 minutes. The OD value at 450 nm was then measured using a microplate reader, and the survival of the cells in each group was compared. In vitro proliferation assays of TetOn-Omomyc inducible expression cell lines and MYC-AID cell lines required 72 hours of culture, and the other steps were the same.

[0087] 3. Experimental results

[0088] Test results such as Figure 7 Middle A, Figure 8 Middle B and Figure 10 As shown, the results showed that targeted degradation of endogenous MYC protein, induction of Omomyc expression or MYC small molecule inhibitors significantly enhanced the proliferation inhibitory ability of BETi on CRC cells, and drug synergy analysis showed that MYCi and BETi exhibited a strong synergistic effect in CRC cells.

[0089] Example 4 CRC cell plate colony formation experiment

[0090] 1. Experimental Materials

[0091] The MYC-AID cell line (HT29-MYC-AID) successfully constructed in Example 1 and the TetOn-Omomyc inducible expression system cell lines (RKO-TetOn-Omomyc, CACO2-TetOn-Omomyc, HT29-TetOn-Omomyc, HCT15-TetOn-Omomyc) successfully constructed in Example 2, RPMI1640 medium, DMEM medium, doxycycline, IAA, BETi (JQ1, ARV-771).

[0092] 2. Experimental methods

[0093] ① The above cells in the logarithmic growth phase were seeded at a certain density (3000 / well) in 12-well plates and divided into four groups (blank control group, DOX group, BETi group, and combination drug group, hereinafter referred to as groups 1-4). One concentration gradient was set for each group, and three replicates were set for each concentration gradient. 2 mL of RPMI1640 medium was added to each well. Groups 2 and 4 were immediately added with doxycycline or IAA (1 μg / mL doxycycline for TetOn-Omomyc inducible expression cell lines and 500 μM IAA for MYC-AID cell lines) and cultured for 24 hours.

[0094] ② Groups 3 and 4 were added with BETi alone at the concentration shown in Table 2 and cultured in an incubator for 1-2 weeks, with doxycycline added every 48 hours to a concentration of 1 μg / mL.

[0095] Table 2 Drug concentrations used in in vitro clone formation experiments

[0096]

[0097] ③ When obvious cell clones are visible on the well wall, stop the culture and perform crystal violet staining.

[0098] ④ After staining, wait for the moisture in the wells to dry, scan the well plate, and perform statistical analysis on the results.

[0099] 3. Experimental results

[0100] Test results such as Figure 7 Middle B, Figure 9 As shown in middle A, the results showed that inducing MYC protein degradation or inducing Omomyc expression significantly enhanced the inhibitory ability of BETi on the clone formation of CRC cells.

[0101] Example 5 CRC cell sphere formation experiment

[0102] 1. Experimental Materials

[0103] The MYC-AID cell line (HT29-MYC-AID) successfully constructed in Example 1 and the TetOn-Omomyc inducible expression system cell lines (RKO-TetOn-Omomyc, CACO2-TetOn-Omomyc, HT29-TetOn-Omomyc, HCT15-TetOn-Omomyc) successfully constructed in Example 2, the spheroid formation culture medium includes: serum-free DMEM / F-12, 20 ng / mL fibroblast growth factor FGF, 20 ng / mL epidermal growth factor EGF, 1×B-27 (Gibco, catalog number: 17504044) and 5 μg / mL insulin, doxycycline, IAA, BETi (JQ1, ARV-771).

[0104] 2. Experimental methods

[0105] ① The above cells in the logarithmic growth phase were seeded at a certain density in low-adhesion 24-well plates and divided into four groups (blank control group, targeted MYC inhibition group, BETi group, and combination group, hereinafter referred to as Groups 1-4). Each group was set up with one concentration gradient, and each concentration gradient was set up with three replicates. 1 mL of spheroid formation medium was added to each well. For in vitro cell spheroid formation experiments with TetOn-Omomyc-inducible expression cell lines and MYC-AID cell lines, doxycycline / IAA was immediately added to Groups 2 and 4 (1 μg / mL doxycycline for TetOn-Omomyc-inducible expression cell lines and 500 μM IAA for MYC-AID cell lines) and cultured for 24 hours.

[0106] ② Groups 3 and 4 were added with BETi at concentrations shown in Table 3 and cultured in an incubator for 5-7 days, with doxycycline added every 48 hours to a concentration of 1 μg / mL.

[0107] Table 3 Drug concentrations used in in vitro spheroidization experiments

[0108]

[0109] ③ After the cell spheroids reach an appropriate size, take photos under a microscope and perform statistical analysis on the spheroid images.

[0110] 3. Experimental results

[0111] Test results such as Figure 7 Middle C, Figure 9 As shown in Figure 2B, the results showed that inducing MYC protein degradation or inducing Omomyc expression significantly enhanced the inhibitory ability of BETi on spheroid formation of CRC cells.

[0112] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Application of MYC inhibitors in the preparation of BET inhibitor sensitizing drugs; The sensitizing drug enhances the tumor therapeutic effect of the BET inhibitor by reducing the binding ability of BRD4 in the super enhancer region; The MYC inhibitor is selected from one or more of the following structures: ； The tumor is colorectal cancer; The BET inhibitor is selected from one or more of the following structures: 。 2. The use according to claim 1, characterized in that The BET inhibitor exerts a tumor therapeutic effect by targeting and binding to BRD4.

3. An antitumor composition, characterized in that: The anti-tumor composition comprises a BET inhibitor and a sensitizing drug, wherein the sensitizing drug comprises a MYC inhibitor; The sensitizing drug enhances the tumor therapeutic effect of the BET inhibitor by reducing the binding ability of BRD4 in the super enhancer region; The MYC inhibitor is selected from one or more of the following structures: ； The tumor is colorectal cancer; The BET inhibitor is selected from one or more of the following structures: 。 4. The antitumor composition according to claim 3, characterized in that The anti-tumor composition further comprises a pharmaceutically acceptable carrier.

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