Application of MYC inhibitor in preparation of BET inhibitor sensitizing drug

Abstract

The invention relates to the technical field of biological medicine, and discloses application of an MYC inhibitor in preparation of a BET inhibitor sensitizing drug. In order to screen a synergistic drug of a BET inhibitor, a core transcription factor for regulating a super enhancer (SE) is systematically screened, and it is found that MYC is a core main transcription factor of a tumor SE regulation network. Furthermore, experiments prove that the cancer inhibition effect of the BET inhibitor can be remarkably improved and the effective concentration of the BET inhibitor can be reduced by inducing and degrading endogenous MYC protein, inducing expression of negative dominant inhibition polypeptide Omomyc of MYC or treating with an MYC small-molecule inhibitor. Therefore, the MYC inhibitor can be used as a BET inhibitor sensitizing drug, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technologies, and particularly to the application of MYC inhibitors in the preparation of drugs for sensitizing BET inhibitors. Background Art

[0002] Among the cis-regulatory elements of eukaryotic transcriptional activity, a class of super-enhancers (SEs) with significantly higher activity than ordinary enhancers have been found to be key regulators of cell fate-determining genes and also control the transcriptional output of many disease-related genes. Compared with the size of ordinary enhancers ranging from hundreds to thousands of base pairs, SEs usually cover a vast region of 10 kb to 20 kb, may contain multiple enhancer clusters inside, and are highly enriched in transcription factors and chromatin regulators. Epigenomic studies in colorectal cancer (CRC), gastric cancer, nasopharyngeal cancer and other epithelial tumors have clearly pointed out that abnormal SE activity is a key factor promoting carcinogenesis. Research shows that genes controlled by SEs are more sensitive to changes in external environmental signals than ordinary genes, so selectively targeting SEs has become a possible option for cancer treatment. Indeed, small molecule drugs (BETi) that target and inhibit the Bromodomain and extraterminal (BET) domain proteins have shown good anti-cancer effects in preclinical studies, and the treatment with these drugs selectively downregulates the expression of oncogenes regulated by SEs. Among them, BET family proteins, including BRD2, BRD3, BRD4, and BRDT, etc., as epigenetic readers, are regarded as key therapeutic targets due to their ability to recognize and bind acetylated histones (such as H3K27ac) and their important role in the activity of SEs. The BET inhibitors (BETi) developed based on this inhibit the tumor process by competitively binding to the BET protein domain and blocking its ability to recognize H3K27ac and transcription factors.

[0003] Although BET inhibitors have shown good application prospects in a large number of preclinical models and multiple clinical trials for solid and blood cancers are underway, they have not been approved due to problems such as high-dose toxicity and drug resistance. For example, GSK3358699 was eliminated due to severe adverse reactions mainly headache at doses lower than the expected therapeutic dose; OTX015 was used to treat acute myeloid leukemia and various solid tumors in a phase I clinical study, but most patients had gastrointestinal symptoms such as anemia, neutropenia, nausea, and diarrhea; iBET762 was used to treat various tumors such as neuroblastoma and pancreatic cancer in preclinical experiments, but it had similar toxic side effects to OTX015 in clinical trials, indicating that these toxicities may be common in BET inhibitors. And now the research optimized for such problems mainly focuses on the combination of drugs, reducing the dose of BET inhibitors by combining synergistic drugs to reduce the occurrence of toxic side effects and obtain better therapeutic effects. Therefore, it is crucial to find a therapeutic strategy that synergizes with BET inhibitors. Summary of the Invention

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

[0005] The technical solution of the present invention is as follows: In the first aspect, there is provided the application of MYC inhibitors in the preparation of BET inhibitor-sensitizing drugs.

[0006] In a preferred technical solution, the sensitizing drug is used to enhance the tumor treatment effect of BET inhibitors.

[0007] In a further preferred technical solution, the tumor is colorectal cancer.

[0008] In a preferred technical solution, the BET inhibitor exerts its tumor treatment effect by targeting and binding to BRD4.

[0009] In a preferred technical solution, the BET inhibitor is selected from one or more of the following structures: .

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

[0011] In a preferred technical solution, the MYC inhibitor is a polypeptide with an amino acid sequence as shown in SEQ ID NO.1.

[0012] In a second aspect, an anti-tumor composition is provided. The anti-tumor composition comprises a BET inhibitor and a sensitizing drug, and the sensitizing drug comprises a MYC inhibitor.

[0013] In a preferred technical solution, the anti-tumor composition further comprises a pharmaceutically acceptable carrier.

[0014] Beneficial effects: The present invention provides the application of a MYC inhibitor in the preparation of a BET inhibitor-sensitizing drug, and for the first time proposes that simultaneous targeted intervention of MYC and BRD4 can synergistically inhibit oncogenic transcription and thus block tumor growth. Experiments have proved that inducing the degradation of endogenous MYC protein, inducing the expression of the dominant-negative inhibitory polypeptide Omomyc of MYC or treating with a MYC small molecule inhibitor can significantly enhance the anti-tumor effect of the BET inhibitor and reduce the effective concentration of the BET inhibitor. Therefore, the MYC inhibitor can be used as a BET inhibitor-sensitizing drug and has good application prospects. Description of the Drawings

[0015] Figure 1 It is a diagram showing the identification results of MYC as the core transcription factor regulating SE activity; wherein, A is the transcription factor motif enrichment profile of SE regions across cancer types, based on the SE motif enrichment analysis of 14 tumor cell lines; B is the occupancy preference of MYC in cis-regulatory elements, and the percentage of cis-elements occupied by MYC in different cancer cell lines (there is at least one MYC ChIP-seq peak in the element region obtained from the same cell line), and each point represents a cell line; C is the co-localization feature of MYC and SE activity markers. After sorting the enhancers based on the H3K27ac signal intensity in K562 cells, the ChIP-seq signal intensities of H3K27ac, MYC, MED1, and BRD4 are visualized.

[0016] Figure 2 It is a diagram showing the analysis results of the preferential enrichment of MYC in tumor type-specific SE; wherein, A is the distribution of each factor on the SE occupied by MYC across cell lines; B is the visualization of ChIP-seq by IGV, showing the enrichment of MYC on the SE of its own locus in different tumor cell types.

[0017] Figure 3 It is a diagram showing the construction and verification results of an endogenous MYC inducible degradation system; wherein, A is a schematic diagram of the construction of MYC-AID protein degradation; B is the protein blot analysis results of the protein cell lysates obtained from the MYC-AID cell lines established in HT29 or LoVo, treated with 500 μM IAA at the specified time points or washed with PBS after 3 hours of IAA treatment; the HA antibody is used to detect the endogenous MYC protein fused with the HA tag, which produces the same results as the MYC antibody; GAPDH is used as a control.

[0018] Figure 4 It is the GSEA result of HT29-MYC-AID cells only targeting the SE gene.

[0019] Figure 5 It is the verification result graph of the co-occupation of MYC and BRD4 in the SE region; among them, A is the schematic diagram of the functional interaction mechanism between MYC and BRD4; B is the result of the co-immunoprecipitation (Co-IP) experiment in HT29-MYC-AID and LoVo-MYC-AID cells, as the verification of the endogenous interaction between MYC and BRD4; C is the correlation scatter plot of the enrichment of MYC and BRD4 on SEs, reflecting the strong correlation between the occupation of MYC and BRD4 in the SE region; D is the binding ratio of MYC and BRD4 in the SE region.

[0020] Figure 6 It is the verification result graph of MYC regulating the enrichment of BRD4 in the SE region; among them, A is the BRD4 ChIP-seq signal in the SE region where MYC occupies or does not occupy; B is the BRD4 ChIP-seq signal in the SE region after MYC degradation induced by IAA in HT29-MYC-AID and LoVo-MYC-AID cells; C is the heat map of the ChIP-seq signal intensity of the indicating factors within a 10kb window centered on the enhancer in HT29-MYC-AID and LoVo-MYC-AID cells.

[0021] Figure 7 It is the result graph of the in vitro proliferation inhibition experiment, colony formation, and spheroid formation experiments of the MYC-AID cell line in Examples 3-5; among them, A is the result of the in vitro proliferation inhibition experiment; B is the result of the colony formation experiment; C is the result of the spheroid formation experiment.

[0022] Figure 8 It is the detection result of the TetOn-Omomyc inducible expression system cell line in Example 2 and the result graph of the in vitro proliferation experiment of the TetOn-Omomyc inducible expression system cell line in Examples 3-5; among them, A is the result of the Western blot analysis of the TetOn-Omomyc inducible expression system cell line; B is the result of the in vitro proliferation inhibition experiment.

[0023] Figure 9 It is the result graph of the in vitro colony formation and spheroid formation experiments of the TetOn-Omomyc inducible expression system cell line in Examples 4-5; among them, A is the result of the colony formation experiment; B is the result of the spheroid formation experiment.

[0024] Figure 10 It is the result graph of the in vitro proliferation experiment of two inhibitors in the human colorectal cancer cell line in Example 3; among them, A is the partial specific result of the synergy of the two inhibitors; B is the statistical result of the average synergy score of the two inhibitors. Detailed implementation mode

[0025] The present invention provides the use of MYC inhibitors in the preparation of drugs for sensitizing BET inhibitors. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention is further described in detail below.

[0026] BETi that targets and inhibits BET family proteins has shown good anti-cancer effects in preclinical studies, and multiple clinical trials for solid and blood type cancers are underway. However, it has not been approved due to problems such as high-dose toxicity and side effects and drug resistance. Therefore, it is crucial to find synergistic drugs to reduce the dose of BETi and thus reduce the toxicity and side effects.

[0027] To screen for synergistic drugs for BETi, the research plan of the present invention is as follows: 1. Identification of MYC as the main transcription factor regulating SE activity To systematically screen for core transcription factors regulating SE, the present invention integrated motif enrichment analysis of SE regions in 14 cell lines (covering five hematological tumors and six solid cancers), and found that MYC was significantly enriched on the SE of all cell lines ( Figure 1 in A). By chromatin immunoprecipitation high-throughput sequencing (ChIP-seq), the occupancy map of MYC in the SE and typical enhancer (TE) regions defined by H3K27ac was constructed. The results showed that the median proportion of the SE regions bound by MYC reached 85% (interquartile range: 56%-100%), and this phenomenon was independent of the cell lineage origin ( Figure 1 in B), suggesting that MYC may be the core main transcription factor of the tumor SE regulatory network.

[0028] To analyze the correlation between MYC and SE activity, the present invention first ranked the whole-genome enhancers based on the H3K27ac signal intensity in K562 cells, and visualized the enrichment signals of MYC and the SE regulatory proteins BRD4 and MED1 corresponding to each enhancer. The enrichment of MYC in the enhancers was highly consistent with the enrichment of H3K27ac, BRD4, and MED1 related to enhancer activity, suggesting that the enrichment of MYC may be another biomarker of active SE ( Figure 1 in C).

[0029] 2. MYC selectively occupies tumor type-specific SE One of the core characteristics of SE is its tissue context specificity, and this dynamic regulatory property makes it a key condition for precisely controlling transcriptional output under specific conditions. Analysis of ChIP-seq results of different types of tumor cell lines showed that MYC selectively occupies tumor type-specific SE ( Figure 2In A), MYC simultaneously binds to the SE region of its own locus in a tumor-specific manner ( Figure 2 In B), indicating the existence of a positive feedback loop for MYC self-regulation.

[0030] 3. Acute degradation of MYC protein in CRC cells selectively inhibits SE-driven transcription To further explore the direct regulatory role of MYC in SE activity, the present invention utilized 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 In A). Its induction and degradation efficiency was verified by Western Blot (WB). The expression of endogenous MYC was almost completely degraded after 0.5 hours of induction by auxin (IAA), and the expression recovered to the baseline level 48 hours after removing IAA ( Figure 3 In B), indicating that this system can achieve time-dependent manipulation of MYC activity.

[0031] Through the auxin-inducible degradation system for MYC protein (MYC-AID), the present invention first classified MYC-regulated genes according to the difference in MYC binding strength between enhancers and promoters, and performed gene set enrichment analysis (GSEA) on different gene sets based on the binding preference of MYC to cis-acting elements. The results showed that compared with other MYC-regulated genes, the gene expression levels controlled by SE with higher MYC binding strength were more sensitive to acute MYC degradation (Table 1), and after MYC recovery, their transcriptional levels rebounded more significantly ( Figure 4 ). These results confirm that MYC specifically regulates tumor cell transcriptional output through SE.

[0032] Table 1 GSEA results of four MYC-regulated gene sets classified according to MYC binding preference

[0033] 4. MYC and BRD4 co-occupy SE active elements in tumor cells BRD4 is one of the known key SE regulators, and the above research revealed the importance of MYC in SE activity regulation, suggesting a potential synergistic effect between the two in SE regulation, that is, their mutual dependence in SE regulation function ( Figure 5 In A), the present invention confirmed the physical interaction between MYC and BRD4 proteins by co-immunoprecipitation (Co-IP) ( Figure 5 In B). Secondly, correlation analysis of the enrichment levels of MYC and BRD4 on SE found a strong positive correlation between the two (Figure 5 In C). In addition, genome-wide chromatin occupancy profiling shows that the binding positions of BRD4 and MYC in cancer cells of different tissue origins are highly consistent genomically ( Figure 5 In D).

[0034] 5. The enrichment of BRD4 in SE regions is partially dependent on MYC In CRC cells, the enrichment of BRD4 in SE elements occupied by MYC is significantly higher than that in SE elements not occupied by MYC ( Figure 6 In A), and acute degradation of MYC leads to a decrease in the binding ability of BRD4 in SE regions ( Figure 6 In B and C), suggesting 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 targeting both of them simultaneously.

[0035] Based on the above research, the embodiments of the present invention provide the use of MYC inhibitors in the preparation of BET inhibitor-sensitizing drugs.

[0036] Specifically, as one of the most representative genes controlled by SE in various cancer types, MYC has long been considered to be "downstream" of the SE regulatory machinery (such as BRD proteins), mainly acting as an effector to amplify the SE transcriptional cascade. At the same time, as a master transcription factor, MYC is crucial for maintaining normal cell proliferation, and when it is overactivated, it may lead to cell carcinogenesis. In CRC, the overactivation of MYC may be due to APC gene mutations or MYC gene copy number amplification. The present invention break throughly reveals that MYC can enrich and bind to tumor type-specific SE and actively regulate SE activity, and there is a functional interdependence between MYC and BRD4 in controlling SE-driven oncogenic transcription, and accordingly proposes that simultaneous targeted intervention of MYC and BRD4 can synergistically inhibit oncogenic transcription and thus block tumor growth. The present invention uses colorectal cancer (CRC) as a model to intervene the activity of MYC in CRC cells through three different ways: inducing the degradation of endogenous MYC protein, inducing the expression of the dominant-negative inhibitory polypeptide Omomyc of MYC, or treating with MYC small molecule inhibitors. The results consistently prove that the decrease in MYC activity can effectively sensitize BET inhibitors. Therefore, MYC inhibitors can be used to prepare BET inhibitor-sensitizing drugs.

[0037] Currently, the approaches for targeted intervention of MYC mainly fall into two categories: one is to use small molecule inhibitors of MYC (MYC inhibitor, MYCi) to interfere with the binding of the MYC / MAX complex and thus exert its effect. For example, MYCi975 can bind to the carboxyl-terminal bHLHZip domain of MYC and disrupt the formation of the MYC / MAX complex. The other category is the small molecule polypeptide Omomyc composed of 91 amino acids (its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.1: MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA, and the PDB ID is pdb_00005i50). As a dominant negative mutant of MYC, Omomyc contains the carboxyl-terminal bHLHZip of MYC, in which the leucine zipper carries four point mutations, namely 63T, 70I, 76Q, and 77N. These mutations change the dimerization specificity of Omomyc, enabling it to bind to MAX in the form of homodimers or heterodimers. This binding allows Omomyc to competitively recognize and bind to the E-box DNA sequence with the MYC / MAX complex. Through this intermolecular competition, Omomyc significantly reduces the binding affinity of the MYC / MAX complex in the promoter region of its target genes, thereby inhibiting the transcriptional activation function mediated by MYC. At the same time, Omomyc has been shown to exhibit anti-cancer activity in various preclinical models. Currently, OMO-103 developed based on Omomyc has fully passed the first-phase clinical trial, showing potential efficacy and acceptable toxicity.

[0038] The present invention will be further described below through specific examples.

[0039] Example 1 Construction of a MYC protein inducible degradation system (AID) in CRC cells 1. Experimental materials Human colorectal cancer cell lines HT29 (MYC gene amplified, APC gene mutant type), LoVo (MYC gene non-amplified, APC gene mutant type), RPMI1640 medium, transfection reagent, plasmid, serum, and double antibody.

[0040] 2. Experimental methods (1) Construction of a stable line overexpressing the auxin receptor F-box protein (AtAFB) ① Co-transfect plasmid P-AtAFB2-mCherry-weak NLS (Addgene, Catalog No.: 129717) and pCas9-sgAAVS1-1 (Addgene, Catalog No.: 129726) into HT29 and LoVo cells respectively.

[0041] ② Screen positive cells with antibiotics, amplify and culture after detecting transfection efficiency, and select monoclonal cells to establish stable cell lines, obtaining HT29 and LoVo cells stably expressing AtAFB2.

[0042] (2) Integrate the degron tag minilAA7 into the target gene MYC ① Design sgRNA at the stop codon position of the target gene.

[0043] ② Clone the sgRNA into the pre-digested lentiCRISPR v2 vector (Addgene, Catalog No.: 52961) to obtain the sgRNA recombinant plasmid.

[0044] ③ Construct a homologous repair plasmid containing the left homologous arm - miniIAA7 and the right homologous arm - Blastidin resistance gene.

[0045] ④ Co-transfect the sgRNA recombinant plasmid and the homologous repair plasmid into HT29 and LoVo cells stably expressing AtAFB2.

[0046] ⑤ Amplify and culture after antibiotic screening and transfection efficiency detection, and perform monoclonal screening to obtain HT29 cells with an MYC protein inducible degradation system (HT29-MYC-AID) and LoVo cells with an MYC protein inducible degradation system (LoVo-MYC-AID).

[0047] ⑥ Extract genomic DNA from HT29-MYC-AID and LoVo-MYC-AID respectively, verify the correct integration of the miniIAA7 sequence by PCR, and perform Western Blot detection to verify its inducible degradation efficiency.

[0048] 3. Experimental Results The detection results are as Figure 3 shown in B below. The WB results show that an MYC protein inducible degradation system was successfully constructed in colorectal cancer cell lines with and without MYC gene amplification, and this system can achieve time-dependent manipulation of MYC activity.

[0049] Example 2 Construction of a TetOn-Omomyc Inducible Expression System in CRC Cells 1. Experimental Materials Human colorectal cancer cell lines HT29 (MYC gene amplified, APC gene mutant), RKO (MYC gene amplified, APC gene wild-type), HCT15 (MYC gene non-amplified, APC gene mutant), CACO2 (MYC gene non-amplified, APC gene mutant), human renal epithelial cell line Lentix-293T (Clonetech, catalog number: 632180), DMEM and RPMI1640 media, PEI transfection reagent, plasmids, serum, and double antibiotics.

[0050] 2. Experimental methods (1) Virus packaging ① One day in advance, inoculate Lentix-293T cells into a 6-well plate and culture them with DMEM complete medium, and the cells need to reach 70%-80% within 1 day.

[0051] ② 30 minutes before transfection, replace the DMEM complete medium in the plate with DMEM medium without antibiotics and serum.

[0052] ③ Add 200 μL of DMEM medium without antibiotics and serum, PEI transfection reagent, packaging plasmids (0.7 μg VSVG (Addgene, catalog number: 8454), 2.1 μg PAX2 (Addgene, catalog number: 12260)), and the target plasmid (3.2 μg pLVX-TetOne-Omomyc (Clontech, catalog number: 631847)) into a 1.5 mL EP tube, gently pipette and mix well, and let it stand at room temperature for 20 minutes to obtain a mixed solution.

[0053] ③ Gently drop the above mixed solution into the culture medium of Lentix-293T cells, place it in a cell incubator for 6-8 hours, replace the culture medium with fresh complete medium, and collect the cell supernatant produced at 48 hours and 72 hours after the medium change, and use them together to obtain the virus supernatant.

[0054] (2) Virus infection ① One day in advance, inoculate the target cells to be infected into a 6-well plate, and perform virus infection after the cell density reaches 40-50%.

[0055] ② After filtering the virus supernatant with a 0.45 μm filter membrane, mix the virus supernatant filtrate and fresh medium at a volume ratio of 3:1 and add 5 μg / mL of polybrene, then add it to the 6-well plate, place it in an incubator for 10-12 hours, and replace the culture medium with fresh complete medium.

[0056] ③ 48 hours after the medium change, perform screening with the corresponding antibiotics and conduct Western Blot detection.

[0057] 3. Experimental Results The detection results are as Figure 8 shown in A below. The WB results show that the TetOn-Omomyc inducible expression system was successfully constructed in colorectal cancer cell lines with and without MYC amplification. This system can express the MYC dominant-negative inhibitory polypeptide Omomyc tagged with ubiquitin protein (HA). The amino acid sequence of Omomyc is shown in SEQ ID NO.1.

[0058] Example 3 CRC Cell Proliferation Inhibition Experiment 1. Experimental Materials Human colorectal cancer cell lines HT29 (MYC gene amplified, APC gene mutant), RKO (MYC gene amplified, APC gene wild-type), HCT15 (MYC gene not amplified, APC gene mutant), CACO2 (MYC gene not amplified, APC gene mutant), the MYC MYC-AID cell lines (HT29-MYC-AID, LOVO-MYC-AID) successfully constructed in Example 1 above, 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, doxycycline (Dox), IAA, BETi (JQ1, ARV-771, OTX015, iBET762), MYCi (MYCi975, KJ-Pyr-9, MYCMI-6).

[0059] 2. Experimental Methods ① Take the above cells in the logarithmic growth phase and seed them in 96-well plates at a certain density. For the in vitro proliferation experiments of the two inhibitors in human colorectal cancer cell lines, step ② can be carried out; the in vitro proliferation experiments of the TetOn-Omomyc inducible expression cell lines and the MYC-AID cell lines need to be divided into two groups, each group has 5 concentration gradients, each concentration gradient has 6 replicates, 100 μL of medium is added to each well, and group 1 immediately adds doxycycline or IAA for induction (1 μg / mL of doxycycline is added to the TetOn-Omomyc inducible expression cell lines, and 500 μM of IAA is added to the MYC-AID cell lines), and culture for 24 hours.

[0060] ② After 24 hours, discard the old medium and add medium containing different drug concentrations. The dosing concentrations for the in vitro proliferation experiments of the two inhibitors in human colorectal cancer cell lines are shown in Figure 10In the in vitro proliferation experiments of the TetOn-Omomyc inducible expression cell line and the MYC-AID cell line, in group 2, BETi was added alone. In group 1, in addition to BETi, doxycycline or IAA was added (1 μg / mL of doxycycline was added to the TetOn-Omomyc inducible expression cell line, and 500 μM of IAA was added to the MYC-AID cell line). The dosing concentrations are shown in Figure 7 A in Figure 8 and B in

[0061] ③ After culturing for 48 hours in the in vitro proliferation experiment of the two inhibitors in human colorectal cancer cell lines, the RPMI1640 medium containing the CCK-8 detection reagent was replaced (CCK-8:RPMI1640 = 1:10), and it was incubated in an incubator at 37 °C for 1 hour and 20 minutes. Then, the OD value at 450 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader, and the survival of cells in each group was compared. For the in vitro proliferation experiments of the TetOn-Omomyc inducible expression cell line and the MYC-AID cell line, it was necessary to culture for 72 hours, and the other steps were the same.

[0062] 3. Experimental results The detection results are as shown in Figure 7 A in Figure 8 and B in Figure 10 The results show that targeting the degradation of endogenous MYC protein, inducing the expression of Omomyc, or MYC small molecule inhibitors all significantly enhance the ability of BETi to inhibit the proliferation of CRC cells, and the drug synergy analysis shows that MYCi and BETi exhibit a strong synergistic effect in CRC cells.

[0063] Example 4 CRC cell plate colony formation experiment 1. Experimental materials The MYC-AID cell line (HT29-MYC-AID) successfully constructed in Example 1 above 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).

[0064] 2. Experimental methods ① The above cells in the logarithmic growth phase were seeded in a 12-well plate at a certain density (3000 cells / well) and divided into four groups (blank control group, DOX group, BETi group, and combined drug group, hereinafter referred to as groups 1-4 respectively). Each group was set with one concentration gradient, and three replicates were set for each concentration gradient. 2 mL of RPMI1640 medium was added to each well. Doxycycline or IAA was immediately added to groups 2 and 4 (1 μg / mL of doxycycline was added to the TetOn-Omomyc inducible expression cell line, and 500 μM of IAA was added to the MYC-AID cell line), and cultured for 24 hours.

[0065] ② BETi was added alone to groups 3 and 4, and its concentration is shown in Table 2. It was placed in an incubator and cultured for 1-2 weeks, and doxycycline was supplemented every 48 hours until its concentration reached 1 μg / mL.

[0066] Table 2 Drug concentrations used in the in vitro colony formation assay

[0067] ③ After obvious cell clone clusters were visible on the well wall with the naked eye, the culture was stopped and crystal violet staining was performed.

[0068] ④ After the staining was completed and the water in the wells was air-dried, the well plate was scanned, and the results were statistically analyzed.

[0069] 3. Experimental results The detection results are as Figure 7 shown in B of Figure 9 and A of

[0070] Example 5 Spheroid formation assay of CRC cells 1. Experimental materials The MYC-AID cell line (HT29-MYC-AID) successfully constructed in Example 1 above 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 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).

[0071] 2. Experimental methods ① The above cells in the logarithmic growth phase were inoculated into a low - adhesion 24 - well plate at a certain density and divided into four groups (blank control group, MYC - targeted inhibition group, BETi group, and combined - drug group, hereinafter referred to as groups 1 - 4 respectively). Each group was set with 1 concentration gradient, and 3 replicates were set for each concentration gradient. 1 mL of sphere - forming medium was added to each well. In the in vitro cell sphere - forming experiments of the TetOn - Omomyc inducible expression cell line and the MYC - AID cell line, doxycycline / IAA (1 μg / mL of doxycycline was added to the TetOn - Omomyc inducible expression cell line, and 500 μM of IAA was added to the MYC - AID cell line) was immediately added to groups 2 and 4 and cultured for 24 hours.

[0072] ② BETi was added alone to groups 3 and 4 at the concentrations shown in Table 3 and placed in an incubator for 5 - 7 days. Among them, doxycycline was replenished to a concentration of 1 μg / mL every 48 hours.

[0073] Table 3 Drug concentrations used in the in vitro sphere - forming experiment

[0074] ③ After the cell spheres reached an appropriate size, they were photographed under a microscope, and statistical analysis was performed on the sphere - forming images.

[0075] 3. Experimental results The detection results are as shown in Figure 7 C in Figure 9 and B in

[0076] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description. All such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. Use of an MYC inhibitor in the preparation of a drug for sensitizing a BET inhibitor.

2. The application according to claim 1, wherein The sensitizing drug is used to enhance the antitumor effect of a BET inhibitor.

3. The application according to claim 2, characterized in that, The tumor is colorectal cancer.

4. The application according to claim 1, wherein The BET inhibitor exerts its antitumor effect by targeting and binding to BRD4.

5. The application according to claim 1, characterized in that The BET inhibitor is selected from one or more of the following structures: 。 6. The application according to claim 1, characterized in that, The MYC inhibitor is selected from one or more of the following structures: 。 7. The application according to claim 1, characterized in that The MYC inhibitor is a polypeptide with an amino acid sequence as shown in SEQ ID NO.

1.

8. An anti-tumor composition, characterized in that, The antitumor composition comprises a BET inhibitor and a sensitizing drug, and the sensitizing drug comprises an MYC inhibitor.

9. The anti-tumor composition according to claim 8, wherein The antitumor composition further comprises a pharmaceutically acceptable carrier.

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