Overexpression of BRD4 Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Background:Bromodomain containing protein 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) protein family, has been implicated in the pathogenesis of a number of hematological and solid tumors. BRD4 is an epigenetic reader that plays a central role in transcriptional regulation, cellular growth control, and cell cycle progression. As a consequence, BET inhibitors elicit anticancer activity in numerous malignant contexts. While deregulation of BRD4 activity has been shown to be essential for the maintenance of AML, its expression profile and prognostic value in AML have not been investigated. Additionally, how BRD4 is regulated in cancer cells remains largely unknown.

Methodology: We therefore evaluated the level of BRD4 expression using Reverse Phase Protein Array (RPPA) technology in marrow and blood samples from 511 newly diagnosed AML patients, and determined a correlation between BRD4 expression and clinical characteristics and overall survival in AML patients. We also correlated expression of BRD4 with 232 other proteins (encompassing signal transduction, metabolism, apoptosis and cellular proliferation pathways) that were included in RPPA. Potential role of molecular chaperone heat shock protein 90 (Hsp90) in regulating BRD4 protein stability was also investigated.

Results: BRD4 expression was similar in peripheral blood and bone marrow and expression was found to be higher at diagnosis than at relapse (P = .035). Compared with normal bone marrow derived CD34+ cells, BRD4 expression in AML was higher in 28% of patients and that higher BRD4 levels significantly predicted shorter overall survival ( P = .002). There was no significant difference in BRD4 expression on the basis of age, sex, or performance status, antecedent malignancies, and previous chemotherapy and/or radiotherapy. BRD4 expression levels were not strongly correlated with French-American-British classification (FAB), although levels were slightly higher in M5 subtype. BRD4 levels were found to be significantly higher in patients with NPM1 mutation ( P = .003) or FLT3-ITD ( P = .057) or between those with both FLT3-D835F and NPM1 mutation ( P = .01). BRD4 levels were not associated with the attainment of complete remission (CR), although rates were somewhat lower for those with high. However, patients with high levels of BRD4 had significantly inferior remission duration (25.9 vs 50 weeks; P = 0.03) compared with those with normal levels of BRD4. The overall survival (OS) of patients with higher levels of BRD4 was significantly inferior compared with those with normal levels of BRD4 (28.3 vs 55.3 weeks; P = .002). Patients with unfavorable cytogenetics and above-normal levels of BRD4 had inferior survival compared with those with normal levels of BRD4 (22.4 vs 38.9 weeks; P = .0043; Figure 3C). BRD4 expression was found to be strongly correlated ( R > 0.2 and P < 0.0001 or 0.00001) either positively (n = 48) or negatively (n = 14) with proteins known to be important in AML. BRD4 expression was positively correlated with various cell proliferation-related proteins (Hsp90, pSTAT5, nucleolin and mTOR), anti-apoptotic protein (Bcl-2), oncoproteins (FLI1, CREB and ELK1), proteins important for stem cell functions (Ash2L and LSD1), hematopoietic transcription factors (ERG and PU.1/Spi1), and Smad proteins, signal transducers for the members of the transforming growth factor-β (TGF-β) superfamily. The stability, activity and function of several mutated, chimeric, and overexpressed signaling proteins crucial for oncogenesis are often maintained by Hsp90. Our study also demonstrates that BRD4 stability is dependent upon Hsp90 activity and pharmacologic inhibition of Hsp90 promoted ubiquitination followed by proteasome-dependent degradation of BRD4 protein.

Conclusions: These findings suggest BRD4 as a prognostic biomarker for unfavorable outcomes and supports the clinical rational for using BRD4 inhibitors in AML. Future studies could identify mechanistic insights into BRD4-associated proteins as biomarkers for response to BET inhibition.