The light-labeled and heavy-labeled cells were lysed, and the nuclear extracts were prepared as described previously (70). enrich disproportionately at a subset of key oncogenic and lineage-specific genes such as and selectively stimulates their expression to drive cellular proliferation in cancers (4, 14, 16C18). Blocking bromodomain binding to acetylated histones with BET inhibitors, including (+)-JQ1 (19) and I-BET (20), specifically down-regulate these oncogenes (4, 16, 17). Dependency of the tumor cells to high-level expression of these oncogenes provides the basis for using BET inhibitors to abrogate BRD4 function for treating these cancers (4, 14, 16, 17). Multiple BET inhibitors have thus joined clinical trials. Early clinical trials have shown promising results, especially for hematological malignancies (21), highlighting the potential of targeting BRD4 in anticancer treatment. However, resistance to BET inhibitors has also emerged (22, 23), revealing the therapeutic limitations of BET inhibitors and the complexity of BRD4 regulation mechanisms. More importantly, we and others have shown that BRD4 also plays an important role in noncancerous systems such as mouse embryonic stem cells, preimplantation embryos, and keratinocyte differentiation (9, 24C26). There are growing concerns regarding the consequences of disrupting BRD4 function in the normal cells by using BET inhibitors (27). Therefore, it is critical to elucidate the molecular mechanisms that regulate BRD4s biological function in both normal and disease settings so that therapeutic interventions can be developed to switch off the oncogenic activity of BRD4 specifically in cancer cells while sparing the normal BRD4 function in healthy cells. NMC is usually a highly lethal tumor typically caused by translocation in half, resulting in the in-frame fusion of BRD4 bromodomains and extraterminal domain name with nearly the entire sequence of the gene (15, 28). NMCs represent the most lethal subset of squamous cell carcinomas (15). They metastasize rapidly and are extremely aggressive; patients have a median survival of <7 mo (15). Translocation fusion oncogene in NMC (15), has been described in pediatric head and neck tumors as well as in lung cancers (29). All NMCs carry an intact locus and simultaneously express and the fusion oncogene (30, 31), providing a unique tumor model to investigate how alteration of BRD4 function by oncogenic mutation leads to cancer. The BRD4-NUT fusion oncoprotein is also tethered to acetylated chromatin by the bromodomains (31, 32). It causes malignancy by blocking NMC differentiation while driving tumor growth (15, DTP348 19). We and others exhibited that BRD4-NUT stimulates BRD4 transcription function to activate specifically the expression of oncogenes such as (28, 30) and (18), which collectively drive the potent NMC transforming activity. However, the molecular mechanisms by which BRD4-NUT modulates BRD4 function to induce such highly aggressive carcinomas remain to be elucidated. In this study, we found that BRD4 is usually hyperphosphorylated in NMC tumors and that this hyperphosphorylation is usually linked to its ability to drive oncogene expression and cellular transformation. We found DTP348 that BRD4 is usually hyperphosphorylated in other BRD4-associated cancers as well. Our study revealed a cellular mechanism that could regulate BRD4s biological function through phosphorylation, which, when dysregulated could lead to oncogenesis. Results BRD4 Is usually Hyperphosphorylated in NMC Tumors. From our previous NMC studies (28, 30, 31), we observed that BRD4 isolated from NMC cells, including HCC2429, 10-15, 14169, and Ty-82 DTP348 cells, ALK7 migrates more slowly in SDS/PAGE than do a number of nonCNMC cells, such as HEK293, C33A, HeLa, U2OS, and A549 cells (Fig. 1gene, our observation indicates that BRD4 has different posttranslational modification(s) in NMC and nonCNMC cells. Therefore, we investigated the BRD4 phosphorylation status in these cells. Whole-cell lysates isolated from both HCC2429 and HEK293 cells were reactive to a phospho-BRD4 antibody, -pS484/488 (Fig. S1and Fig. S1and affinity purified on IgG beads. The BRD4-TII beads were incubated with an equal amount of nuclear proteins isolated from HCC2429 or HEK293 cells to immunoprecipitate the kinases for BRD4. The immunocomplexes captured around the beads were then subjected to an in vitro kinase assay (Fig. 2was affinity purified on IgG beads. The beads were either kept in buffer or coimmunoprecipitated with equal amounts of nuclear proteins from HCC2429 or HEK293 cells. After washing, beads were subjected to kinase assay. BRD4-TII.