Bone marrow samples were collected from patients with MDS (n = 58), AML with MDS-related changes AML (AML-MRC) (n = 16), de novo AML (n = 34), and healthy donors (n = 24). All patients included in the study were untreated at the time of sample collection. MDS patients were classified according to 2016 World Health Organization (WHO) classification (14) and according to revised international prognostic staging system (R-IPSS) (15). The cytogenetic risk for MDS and AML was defined according to R-IPSS (15) and to the Medical Research Council cytogenetic classifications (16), respectively. Healthy donors' and patients' characteristics are described in Table 1. All healthy donors and patients signed informed consent forms under a local research protocol. This study was approved by the Institutional Ethical Review Board in accordance to the Helsinki Declaration.

Bone marrow mononuclear cells were isolated by Ficoll-Paque (GE, Uppsala, Sweden) from diagnostic samples of AML patients and cord blood units (CBU) samples from full-term deliveries. After that, primary human CD34⁺ hematopoietic stem and progenitor cells (CD34⁺ cells) were selected using immunomagnetic activated cell sorting columns (Miltenyi Biotech, Auburn, CA, USA), obtaining purity of at least 90%.

A panel of human myeloid leukemia cell lines (HEL, HL60, K562, and U937) was cultured in Roswell Park Memorial Institute medium-1640 (RPMI) (Sigma) containing 10% FBS, glutamine (2 mM), penicillin (100 μg/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL). The cells were maintained in a humidified atmosphere at 37°C and 5% CO₂ and the experiments were performed when they reached exponential growth.

A BRD4 specific inhibitor (JQ1) was kindly provided by Dr. James Bradner (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). The compound was diluted in dimethylsulfoxide (DMSO) to a 10 mM stock solution and was added to the cells at final concentrations of 0.24 μM to 100 μM for 48 h. Azacitidine (AZA) was also diluted in DMSO to a 100 mM stock concentration and added to cells at final concentrations of 1–3 μM for 48 h. The AZA concentration range was chosen based on the established AZA treatment schedule for MDS patients (75 mg/m²/day), which achieved plasma levels in the range of 3–11 μM (17, 18). The dose of 1 μM was used for both drugs when JQ1 and azacitidine were combined. AZD6738 was obtained from Selleck Chemicals (Houston, TX) diluted in DMSO and added to the cells at final concentrations of 1–20 μM. GI₃₀ (drug inhibition of 30%) was determined after 48 h of exposure and used in combination with 1 μM of JQ1 or azacitidine.

RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA, USA) and reversely transcribed into cDNA with the RevertAid H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). The quantitative RT-PCR (qRT-PCR) reaction was run with SYBR Green Master Mix PCR (Fermentas) using the ABI 7500 Sequence Detection System (Applied-Biosystem, Foster City, CA, USA). The values of the relative quantification of gene expression was calculated through the equation 2^−ΔΔCT (19). A negative no “template control” was included for each primer pair and the amplification specificity was verified using a dissociation curve at the end of each run. Three replicas were run on the same plate for each sample. Sense and antisense primers were designed to be complementary to the sequences contained in different exons. The following primers were used: BRD4 long variant (BRD4L), 5′- AAAGGACCTGAAAATCAAGAACATG-3′ and 5′-GAAGCTGTCGCTGGATGACTT-3′; BRD4 short variant (BRD4S) 5′-CTGACAGCGAAGACTCCGAAA-3′ and 5′-GCTATAGCTTGCTGGGAAGGAA-3′, hypoxanthine phosphoribosyltransferase (HPRT), 5′-GAACGTCTTGCTCGAGATGTGA-3′ and 5′-TCCAGCAGGTCAGCAAAGAAT-3′.

Equal amounts of protein were submitted to electrophoresis on SDS polyacrylamide gels under reducing conditions and the nitrocellulose membrane was blotted with specific antibodies. Polyclonal antibodies against cleaved PARP-1 (sc-56196), CDK6 (sc-56282), GAPDH (sc-32233), and ACTIN (sc-1616) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-histone H2AX (Ser139) (pH2AX) (9718), P53 (2524), and anti-caspase 3 (9665) were purchased from Cell signaling (Danvers, MA, USA) and P21 were obtained from Abcam (Cambridge, MA, USA). The target proteins were analyzed by chemiluminescence using an ECL Plus Kit (GE-Healthcare, Buckinghamshire, England). Quantitative analyses of the optical intensities of protein bands were determined with UN-SCAN-IT graph digitalizing software (Silk scientific, UT, USA) and normalized by actin or GAPDH for protein expression.

Cell growth was measured by methylthiazoletetrazolium (MTT) assay. Briefly, 2.5 × 10⁴ cells per well were plated in 96–well plates in RPMI/10% FBS for 48 h. MTT solution (5 mg/mL) was added to each well and incubated at 37°C for 4 h. The reaction was stopped by 0.1 N HCl in anhydrous isopropanol. Cell growth was evaluated by measuring the absorbance at 570 nm, using an automated plate reader (Multiscan MS, Labsystems).

Cell death was measured by annexin-V and PI assay. Briefly, the cells were seeded in 24-well plates and treated or not with different concentrations of JQ1 and/or azacitidine for 48 h. After this period, the cells were collected and incubated with 1 μg/mL propidium iodide (PI) and 1 μg/mL APC-Annexin-V for 15 min at room temperature in the dark. All specimens were analyzed on a FACSCalibur (BD Biosciences, CA, USA) and 10,000 events were acquired for each sample.

U937 cells were transduced with lentivirus-mediated shRNA non-specific control (sc-108080) or lentivirus-mediated shRNA targeting BRD4 (sc-43639-V) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and named shControl and shBRD4 cells, respectively. Pooling of multiple BRD4 shRNAs (three different sequences) was used to reduce possible off-target effects of the shRNAs. BA/F3 cells were transduced with lentivirus particles produced using pCDH-MSV-MCS-EF1α empty vector (control) or containing the full lengths of BRD4S or BRD4L. Briefly, 2 × 10⁵ cells were transduced with lentivirus by spinoculation at multiplicity of infection (MOI) equal to 1 and selected with specific antibiotics for at least 7 days before using in the experiments. BA/F3 cells were also sorted using a FACsAria Fusion (Becton–Dickinson Biosciences) in addition to antibiotic selection.

Cells were seeded on a 24-well plate and treated or not with different concentrations of JQ1 for 48 h. Next, the cells were collected and fixed overnight in 70% ethanol. DNA was stained with Pipes buffer containing PI (20 μg/mL) and RNase A (10 μg/mL). Cell fluorescence was detected with a FACSCalibur. The proportions of cells in the cell cycle phases were analyzed by Modifit (Verify Software House Inc., Topsham, ME, USA), according to DNA distributions.

Statistical analysis was performed using GraphPad Prism5 software (GraphPad Software, Inc., San Diego, CA, USA) or SAS System for Windons 9.2 (SAS Institute, Inc., Cary, NC, USA). Data were expressed as the median [minimum-maximum]. For comparisons, an appropriate Mann–Whitney test, Student's t-test or ANOVA was used. Comparison between patient groups was performed by analysis of t-test and covariance (ANCOVA) controlled for age, followed by post-hoc comparisons using the Tukey test. All experiments were repeated at least four times. Cox regression model was used to estimate overall survival (OS) and event-free survival (EFS) for MDS patients. The stepwise process of selection was used for multivariate analysis. OS was defined as the time (in months) between the date of sampling and the date of death (for deceased patients) or last follow-up (for censored patients). EFS was defined as the time (in months) between the date of sampling and the first event (death or MDS progression or leukemic transformation) or last follow-up (for censored patients). All tests were two-tailed. P ≤ 0.05 were considered statistically significant.

The first step of this study comprised the evaluation of mRNA levels of both BRD4 variants in total bone marrow cells from healthy donors (n = 24), MDS (n = 58), and AML (n = 50) patients. In order to exclude confounders, we carried out an ANCOVA analysis, which showed that age and gender did not interfere in our results.

BRD4S expression was significantly increased in both MDS (4.21 [0.01–56.17]) and AML (4.01 [0.33–26.58]) patients, when compared to healthy donors (2.11 [0.04–10.32]; all P < 0.01) (Figure 1A). No difference in BRD4L expression was observed between healthy donors, MDS and AML patients (Figure 1B). There were no differences when MDS patients were stratified according BM blasts or when AML patients were grouped into de novo AML or AML with myelodysplasia related changes (AML-MRC).

With a median follow-up time of 34.4 months, BRD4S expression appeared as one of the variables with significant impact in event-free survival (EFS) and overall survival (OS) of MDS patients in univariate analysis. The bone marrow blast percentages (absolute values) and R-IPSS (stratified into very low/low, intermediate and high/very high) also significantly altered EFS and OS. Multivariate analysis demonstrated that higher BRD4S expression, along with intermediate and high/very high R-IPSS risk MDS, were the only independent factors related to worse outcomes (Table 2).

Aiming to explore a possible function of BRD4 as an oncogene, we overexpressed BRD4L or BRD4S using GFP tagged in normal hematopoietic murine cells (BA/F3), which are dependent on IL-3, and accessed daily the cell growth for 144 h in the presence or absence of 20 ηg/mL IL-3. The overexpression of both variants was effective (Figures 1C,D), but overexpression of BRD4L or BRD4S did not lead to IL-3 independency (Figure 1E). In the presence of IL-3, the growth of BRD4S or BRD4L overexpressed cells were also similar to the control (data not shown). These results suggest that BRD4 does not act as an oncogene alone.

In order to characterize the role of BRD4 in the disease pathogenesis, a panel of myeloid leukemia cell lines was treated with JQ1, an inhibitor of both BRD4 isoforms and other BET protein members (20, 21). Treatment with JQ1 induced a dose-dependent reduction in cell viability, in association with a similar induction of apoptosis. U937 and K562 cells were more resistant to the treatment, and presented higher GI₅₀ (growth inhibitory concentration, with 50% reduction in cell viability) levels compared with HL60 and HEL cells (Figure 2A). Normal CD34⁺ cells from four samples of cord blood units exhibited greater tolerability to JQ1, with sustained cell viability and lower apoptosis rate compared to neoplastic cells (Figure 2B). JQ1 treatment induced an increase in G0/G1 cell cycle in cell lines, indicating cell cycle arrest (Figure 2C). We also observed an increase in p-H2AX in both HEL (more sensitive) and U937 (more resistant) cell lines, along with decreasing levels of CDK6 and with higher P53 and P21 protein concentrations (Figure 2D). This result suggests that BRD4 pharmacological inhibition causes DDR pathway activation and cell cycle arrest, even when the cells were exposed to a lower dose of JQ1.

With the aim of further characterizing the potential combinatory effects of BRD4 inhibition and a standard treatment for MDS, HEL, and U937 cells were treated with JQ1 and azacitidine for 48 h. JQ1 + AZA treatment increased the apoptotic rate of both cell lines, particularly in U937 cells (Figure 3A). JQ1 treatment increased p-H2AX and cleaved PARP-1, suggesting that activation of DDR is an important mechanism for apoptotic induction after JQ1 treatment. Although annexin V positive cells were increased with the co-treatment, we did not observe the same synergism in pH2AX and cleaved PARP-1 expression, possibly because high levels of these proteins were already detected when JQ1 was used alone (Figure 3B). CD34⁺ cells were isolated from diagnostic samples of 5 AML patients and treated with JQ1, AZA or both. In all samples, the combination of JQ1 and AZA induced a higher apoptosis rate than JQ1 or AZA alone (Figure 3C).

We next sought to investigate whether BRD4 gene silencing would produce similar results to that of JQ1 treatment, in order to exclude possible therapy-related off target effects. For this purpose, U937 cells were stably transduced with lentivirus-mediated shRNA targeting BRD4 (shBRD4) or an appropriate control (shControl) and the efficacy of the transduction was confirmed by qRT-PCR (Figure 4A).

Apoptosis was evaluated in silenced cells treated or not with 1 or 3 μM AZA for 48 h. Following AZA treatment, shBRD4 cells showed a significant increase in apoptotic rate (P < 0.05) (Figures 4B,C), similarly to that observed for the drug combination.

Ataxia telangiectasia and Rad3 related (ATR) inhibitors have been reported to display synergism with JQ1 in the induction of apoptosis in lymphoma and melanoma cells (22, 23). Therefore, we aimed to test and compare the effects of JQ1 combined with a specific ATR inhibitor (AZD6738) or with azacitidine in leukemia cells. The GI₃₀ of AZD6738 was firstly calculated in HEL, U937, and HL60 cell lines (Figure S1). Subsequently, these cell lines were exposed to 1 μM JQ1, 1 μM azacitidine or the GI₃₀ dose of AZD6738 monotherapy or double combined treatment courses (JQ1 + azacitidine, JQ1 + AZD8768, azacitidine + AZD6738) for 48 h. In addition to U937 and HEL cells, a synergic effect of JQ1 and azacitidine was observed in HL60 cells (Figures 5A–F). In HL60 cells, the effects of JQ1 + AZD6738 were stronger than JQ1 + azacitidine (Figure 5A), whereas JQ1 + azacitidine was more potent in U937 cells (Figure 5C) and both combinations produced similar effects in HEL cells (Figure 5E). AZA + AZD6738 was also a promising combination, especially in U937 cells (Figure 5B). pH2AX was induced by all treatment schemes (monotherapy or combinations) in the three cell lines (Figures 5G–I). Cleaved caspase-3 expression was consistent with annexin V and cleaved PARP-1 results (Figures 5G–I).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.