NB4, MV4-11, Kasumi, and THP-1 cells were purchased from National Collection of Authenticated Cell Cultures and were grown in culture in RPMI 1640 medium (GIBCO, Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate at 37°C in 5% CO₂.

Public expressions data of BRD4 in 1389 cell lines was downloaded from DepMap (https://depmap.org/portal/) and shown as a box plot by different tissue/organ. Survival curves of adult patients with AML from TCGA with high/low expression of BRD4/c-MYC were generated from GEPIA2 (http://gepia2.cancer-pku.cn/#index).

AML cell lines were plated at a density of 2 × 10⁴ cells per well in 96-well plate and treated with variable concentrations of dBET1(cat. HY-101838; MedChemExpress). After 48 h of treatment, cell viability was tested with Cell Counting Kit-8 (CCK8) kit (Dojindo Molecular Technologies). Absorbance at 450 nm was applied to estimate the cell viability, which was normalized to DMSO control. Cell viability assay was performed in triplicate, and each assay was repeated at least three times. The half-maximal inhibitory concentration (IC₅₀) of dBET1 was calculated using Graph Prism version 8 (GraphPad Software, Inc., San Diego, CA, United States).

NB4, MV4-11, Kasumi, and THP-1 cells were seeded with the same density (4 × 10³ cells per well) in six-well plate of soft agar supplemented with variable concentrations of dBET1. After 10 days, cells were first fixed with 100% methanol for 15 min, followed by Giemsa staining for 1 h. Images were acquired using an optical microscope, and the number of colonies were manually counted.

Short hairpin RNA (shRNA) targeting CRBN (the sequences: CCG​GGC​CCA​CGA​ATA​GTT​GTC​ATT​TCT​CGA​GAA​ATG​ACA​ACT​ATT​CG) was cloned into pLKO.1 plasmid [30], and cDNA sequence of CRBN tagged with V5 was cloned into pLX304 plasmid [31] (a gift from Dr. X. Liang at the Cancer Science Institute of Singapore). pMD2.G and psPAX2 were used for packaging lentivirus in 293T cells. Twenty hours after incubation with virus supernatant, cells were subjected to puromycin (Cat.#ST551, Beyotime, China) selection (10 μg/ml).

Total RNA was extracted using the RNeasy Mini kit (cat. 74104; Qiagen, Germany). Library preparation, transcriptome sequencing (Illumina NovaSeq 6000) and clean data filtering were carried out by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The 150 bp paired-end reads were aligned to hg38 (Ensembl) genome using HISAT2 software (version 2.2.0). Transcriptome assembly and abundance analysis were performed with StringTie software (version 2.1.2). Differentially expressed genes were identified by R/Bioconductor package DESeq2 with adjusted p value <0.05 and absolute log2 (fold change) > 1. Gene Ontology (GO) and gene set enrichment analysis (GSEA) with Hallmarks gene sets (version 7.1) were conducted using R/Bioconductor package cluster Profiler [32]. Heatmaps of genes were generated by R/Bioconductor package pheatmap. RNA-seq data supporting the results of current study have been deposited in GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE196693). The data can be accessible with a token upon request.

NB4, MV4-11, Kasumi, and THP-1 cells were treated with dBET1 for 24 h and were then fixed with 70% ethanol and incubated at 4°C for overnight, followed by washing with cold 1× phosphate-buffered saline (PBS) buffer. Fixed cells were then incubated with 1.5 μM propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, United States) solution supplemented with RNase A (25 μg/ml) at room temperature for 1 h. Cell cycle distribution was determined using flow cytometry and analyzed using MultiCycle AV DNA analysis software (Verity Software House, Topsham, ME, United States).

Cell apoptosis was analyzed as previously described [33]. Briefly, NB4, MV4-11, Kasumi, and THP-1 cells were treated with dBET1 at indicated concentrations. After 24 h incubation, cells were harvested and washed with cold 1 × PBS buffer. Cells were then suspended in the 1× binding buffer and stained with FITC-Annexin V antibody and PI solution according to the manual of the FITC-Annexin V apoptosis kit (cat. 556420; BD Biosciences, Franklin Lakes, NJ, United States). Cell apoptosis was analyzed by Beckman Gallios™ Flow Cytometer (Beckman, Krefeld, Germany).

Total RNA was purified using the RNeasy Mini Kit (cat. 74104; Qiagen, Germany). First-strand cDNA was generated using a mix of 2 μg of total RNA, 500 ng of random primers (Promega, United States), 200U of M-MLV reverse transcriptase (Promega, United States), and 20U of RNase inhibitor (Thermo Fisher Scientific, MA, United States) in a total volume of 20 μl. qRT-PCR was performed using LightCycler® 480 SYBR Green I Master mix (cat. 04707516001; Roche, Penzberg, Germany) on a Light cycler 480 Real-Time System (Roche, Penzberg, Germany) according to the standard protocol. Quantitative mRNA expression of MYC (forward: ATG​GCC​CAT​TAC​AAA​GCC​G, reverse: TTT​CTG​GAG​TAG​CAG​CTC​CTA​A) was calculated using the Ct method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression (forward: TGC​ACC​ACC​AAC​TGC​TTA​G, reverse: GAT​GCA​GGG​ATG​ATG​TTC) as an internal reference.

Cells were lysed using RIPA buffer containing protease and phosphatase inhibitor cocktail (Roche, Penzberg, Germany) for 30 min on ice. The supernatant was collected by centrifugation as total protein and the concentration of total protein was quantified using the Pierce BCA Kit (Thermo Fisher Scientific). Blotting was performed as previously described [33]. Primary antibodies against the following proteins were used: BRD2(cat:5848s, 1:1,000, Cell Signaling Technology), BRD3(cat: 11859-1-AP, 1:1,000, Proteintech), BRD4 (cat: 13440s, 1:1,000, Cell Signaling Technology), CRBN (cat: HPA045910, Sigma-Aldrich), c-MYC (cat: 9402, 1:1,000, Cell Signaling Technology), PARP (Cat: 9542, 1:1,000, Cell Signaling Technology). GAPDH (1:2,000; MA3374, Millipore) was used as an internal control. The horseradish peroxidase (HRP)-conjugated secondary antibodies: Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L) (cat: 115-035-003) and Goat Anti-Rabbit IgG(H+L) (cat: 111-035-003) were purchased from Jackson ImmunoResearch Laboratories, INC. The bands were visualized using an enhanced chemiluminescent (ECL) detection kit (Pierce, Rockford, IL, United States) using LAS 4010 (GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Various concentrations (0, 0.5, 1.0 μM) of (R)-MG132 (cat.M8699, Sigma-Aldrich, Germany) was used to inhibit proteasome activity in order to show the proteasome-mediated degradation of BRD4, BRD2, and BRD3 by dBET1. For quantified results shown in Figure 1, densities of protein bands of BRD4 in each cell line were quantified by ImageJ and normalized to the densities of GAPDH bands. Quantified densities of BRD4 were then normalized to that of Kasumi cells and used to represent the relative expression of BRD4.

All experiments were performed in triplicates and independently repeated at least three times. Statistical analyses were performed using GraphPad Prism (version 8) (GraphPad Software, Inc., San Diego, CA, United States). p values were estimated by two-tail t test and <0.05 was regarded as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Error bar was indicated by Means ± Standard Deviation (SD).

Recently, our lab [34] reported that high expression of BRD4 in T-ALL cell lines in the Cancer Cell Line Encyclopedia database was associated with poor prognosis of patients with T-ALL in The Cancer Genome Atlas (TCGA) database. Of note, BRD4 was also highly expressed in AML, T-ALL, B-ALL, and B cell lymphoma compared with that in other cancer cell lines (Figure 1A). Taken together, these results signify strong dependence of these cancer cell lines on high expression of BRD4, indicating the potential therapeutic effect of BET family inhibition. Moreover, we explored BRD4 expression in a cohort of 148 adult patients with AML from TCGA database. Patients with AML with high expression of BRD4 have a significantly lower overall survival (OS) rate than those with low expression of BRD4 (p = 0.044) (Figure 1B).

Given its expression in AML cell lines, we hypothesize that survival of AML cell lines might rely on the expression of BRD4 and inhibiting BRD4 might be toxic to AML cell lines. To this end, we first confirmed the expression of BRD4 in four AML cell lines (i.e., NB4, Kasumi, THP-1, and MV4-11) using Western blotting (Figure 1C). BRD4 was expressed in all four cell lines, and its quantified expression is shown in Figure 1D. In addition, the expressions of BRD3 and BRD2 were also confirmed in the same four cell lines (Figure 1E). Then, we performed CCK-8 assay using dBET1, a PROTAC inhibitor targeting BET family to test the cytotoxic effects of inhibiting BET family. Potent cytotoxicity was observed in all four AML cell lines (Kasumi IC₅₀: 0.1483 μM, NB4 IC₅₀: 0.3357 μM, THP-1 IC₅₀: 0.3551 μM, and MV4-11 IC50: 0.2748 μM) (Figure 1F).

To prove that dBET1-mediated degradation of the BRD family (i.e., BRD2, BRD3, and BRD4) has cytotoxic effects on AML cell lines, we determined the protein levels of the BRD family in four AML cell lines treated with increasing concentrations of dBET1. Efficient degradation of the BRD family in a dose-dependent manner was observed in all four cell lines, with the most efficiency observed in Kasumi cells, followed by MV4-11, NB4, and THP-1, which conforms to the IC₅₀ values of dBET1 in these four cell lines: Kasumi (0.1483 μM), followed by MV4-11 (0.2748 μM), NB4 (0.3357 μM), and THP-1 (0.3551 μM) (Figure 2A). Besides, concomitant increasing cleavage of PARP was observed in a dose-dependent manner (Figure 2A, bottom panel). Since cereblon (CRBN) acts as an adaptor between BRD family proteins and dBET1, CRBN depletion would compromise the cytotoxic effects of dBET1. To test this hypothesis, we knocked down CRBN using shRNA (Figure 2B, left panel) in NB4 and MV4-11 cells, followed by CCK-8 assay (Figure 2B, right panel). Compared with the scramble control, AML cell lines with knocked down CRBN were significantly more resistant to dBET1; conversely, restoring CRBN expression by CRBN overexpression in AML cell lines with downregulated CRBN reversed the resistance of AML cell lines to dBET1 (Figure 2B, lower panel). Furthermore, to confirm that BRD family proteins are degraded through proteasome-mediated protein degradation, dBET1 treated AML cell lines were maintained with or without the proteasome inhibitor, (R)-MG132. (R)-MG132 sufficiently repressed the degradation of BRD family proteins in a dose-dependent manner (Figure 2C).

To characterize the effects of dBET1 on cell proliferation, we conducted soft agar colony formation assay with incremental dosages of dBET1. The numbers of colonies significantly decreased after treatment with dBET1 in a dosage-dependent manner in the four AML cell lines (i.e., NB4, Kasumi, THP-1, and MV4-11) (Figure 3A, left panel). The quantified results were shown in the right panel of Figure 3A. Furthermore, cell proliferation was significantly compromised when treated with dBET1 (1 μM) in these four AML cell lines, compared with those treated with DMSO (Figure 3B). These results indicated that dBET1 significantly inhibited cell proliferation of AML cell lines.

To further define the effects of dBET1 on cell proliferation, we first assessed cell cycle distribution after dBET1 treatment. Treatment with dBET1 significantly decreased the percentage of cells in the S phase compared with that treated with DMSO control in a dosage-dependent manner. In contrast, the percentage of cells in the GO/G1 phase significantly increased after dBET1 treatment. Collectively, these results showed that inhibiting BET family by dBET1 arrested the cell cycle of the four AML cell lines tested. Moreover, we performed flow cytometry using Annexin V and PI to detect apoptosis at 24 h after dBET1 treatment (1 μM and 8 μM) in the four AML cell lines. Treatment with dBET1 induced more apoptotic cells in a dosage-dependent manner (Figure 4B, left panel). The quantified percentages of apoptotic cells are shown in the right panel of Figure 4B. Collectively, these results suggested that dBET1 led to arrested the cell cycle and enhanced apoptosis in AML cell lines.

The BRD family plays important roles as a modulator of chromatin remodeling and transcriptional regulation of critical genes in cellular homeostasis and disease progression [35]. Thus, looking into the downstream signaling of the BRD family might provide valuable insights into the underlying mechanism, offering new opportunities for developing better therapies. For this purpose, we compared the transcriptomes of NB4 cells treated with or without dBET1. As a result, 4,409 genes were downregulated, whereas 3,210 genes were upregulated after treatment with dBET1 (Figure 5A volcano plot; Supplementary Data Sheet S1). Then, we performed GO analysis, and apoptosis and cell cycle were among the top enriched biological processes (Figure 5B), which is consistent with the arrested cell cycle and enhanced apoptosis after treatment with dBET1 as shown above. Moreover, downregulated c-MYC target genes were enriched by GSEA (Figure 5C; Supplementary Data Sheet S2). The roles of c-MYC in AML have been extensively studied, and its downregulation would lead to cell cycle arrest and induce apoptosis in AML cell lines [36, 37]. As expected, the expression of c-MYC was downregulated after treatment of dBET1 in a dose-dependent manner in the four AML cell lines, determined by Western blotting and qRT-PCR (Figures 5D,E). In addition to c-MYC, its target genes were also downregulated after dBET1 treatment (Figure 5F). Furthermore, high expression of c-MYC was significantly associated with poor prognosis in a cohort of 148 adult patients with AML from TCGA database (Figure 5G). Taken together, these results highlighted c-MYC as a downstream effector of dBET1 in AML cell lines.