BRD4 Short Isoform Is a Myelodysplasia Prognostic Marker Involved in Inappropriate DNA Damage Response

INTRODUCTION: Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. BRD4 is a BET member of bromodomain-containing proteins, known as epigenome readers, exerting key roles in chromatin remodeling and transcriptional regulation. BRD4 gene encodes two major isoforms, short (BRD4S) and long (BRD4L). Recently, Floyd SR et al (Nature, 2013) described BRD4S as an endogenous inhibitor of the DNA damage response (DDR). Under normal conditions, damaged DNA induces histone phosphorylation of H2AX at Ser 139 (γ-H2AX) and activates a protective signaling network that blocks cell cycle and recruits DNA repair factors. Despite having been described as a therapeutic target in acute myeloid leukemia (AML) by Zuber J et al (Nature, 2011), BRD4 has never been studied in myelodysplastic syndrome (MDS) and its role in the pathogenesis is currently unknown.

AIMS: To evaluate BRD4L and BRD4S expression in MDS and AML patients, correlating with clinical data, progression and survival. We also explored the BRD4 role in DDR signaling using human leukemia cell line models.

MATERIAL AND METHODS: Diagnostic total bone marrow (BM) samples from 24 healthy donors (HD) and 99 patients, including 48 MDS (22 higher-risk MDS and 36 lower-risk MDS) and 51 AML (16 of them with MDS-related changes, AML-MRC), were collected. We also isolated CD34⁺ cells from 7 HD, 5 de novo AML, 4 AML-MRC and 14 MDS (6 higher-risk and 8 lower-risk MDS). BRD4L and BRD4S gene expressions were assessed through q-PCR and expressed as median (minimum-maximum). MDS patients were stratified according IPSS, WHO classification, R-IPSS and cytogenetic risk. Samples were age-adjusted when significant differences were observed, using ANOVA and Tukey’s test. Progression-free and overall survival curves were estimated by Kaplan-Meier method and were analyzed by the Wilcoxon´s test and Cox regression. JQ1, a specific BRD4 inhibitor, was kindly provided by James Bradner. A panel of human myeloid leukemia cell lines (KG1a, HEL, HL60, U937) in exponential growth was treated with increasing doses of JQ1 for 48 hours and cell growth (MTT colorimetric assay), apoptosis (annexin-V/PI) and cell cycle (flow cytofluorometric analysis detecting nuclear PI incorporation) were evaluated. We also determined the expression of p-γH2AX (DDR signaling) by western blot, after 12 hours of JQ1 treatment.

RESULTS: A higher expression of BRD4S was observed in total BM cells from AML (4.01 [0.33-2.58], P=.01) and MDS patients (4.21 [0.01-56.17], P=.01) compared with HD (2.11 [0.04-10.32], P=.01). When stratified according WHO classification, AML-MRC (4.5 [0.33-25.22], P=.04) and higher-risk MDS (4.66 [0.17-56.17], P=.04) subgroups showed higher BRD4S expression. In CD34⁺ cells, BRD4S expression was increased in de novo AML (0.28 [0.21-0.45]) compared with lower-risk MDS (0.02 [0.00-0.44], P=.01). BRD4L mRNA expression was not modulated in total BM and CD34⁺ cells from any subgroup. With median follow-up time of 34.4 months, we found that higher BRD4S gene expression was a worse prognostic factor for MDS transformation and survival, along with IPSS, R-IPSS, low hemoglobin (less than 10g/dL) and higher BM blast percentage. After multivariate analysis, BRD4S gene expression and higher-risk (very high, high and intermediate) R-IPSS remained as independent prognostic factors for MDS progression and overall survival. KG1a and U937 cells showed greater resistance to JQ1, with lower apoptosis rate and proliferation (IC50 not reached, over 3000nM), whereas HEL and HL60 were more responsive (IC50 under 800nM in both cell lines). JQ1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and apoptosis and caused a progressive increase in histone phosphorylation of γ-H2AX, indicating activation of DDR signaling.

CONCLUSIONS: MDS is a clonal myeloid neoplasm characterized by profound epigenetic modifications. Our data establishes BRD4S as a novel MDS prognostic factor, related to aggressive phenotype, higher progression rate and shorter survival. Biologically, BRD4S plays a role in the inappropriate DNA Damage Response of MDS, favoring the disease towards genetic instability and clonal evolution.