32-4600); ATR (Cell Signaling Technology, Catalog No. despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication Deramciclane conflicts, DNA damage, and cell death in oncogenic cells. promoter in oncogenic cells, making it an attractive target in multiple models of cancer19,20. We previously reported a novel role for BRD4 in insulating the chromatin against radiation-induced DNA damage response signaling in oncogenic cells21. In the course of that study, we observed separately that in Deramciclane some cell types, BET bromodomain protein inhibition led to increased DNA damage signaling even in the absence of exogenous DNA damage sources. We noted that cell types with robust DNA damage responses to BET bromodomain inhibition alone were frequently oncogene-driven and rapidly proliferating, leading us to hypothesize that the mechanism of DNA damage involved both replication and the known role of BET bromodomain proteins in transcriptional regulation. Here, we report that BRD4 loss of function leads to the accumulation of R-loops in oncogenic cells causing increased transcriptionCreplication collision events, DNA DSB formation, DNA damage response signaling, and apoptosis. R-loop-induced DNA damage could be reversed by overexpression of RNase H1 or by inhibiting the initiation of transcription using triptolide. These findings reveal the importance of BRD4 in preventing TRCs and regulating?DNA damage checkpoint signaling in oncogenic cells. Results BRD4 bromodomain inhibition causes DNA damage and apoptosis To further explore our previous finding that BRD4 is involved in regulating the DNA damage response in oncogenic cells21, we treated cells with the prototypical BET bromodomain inhibitor JQ122 and assayed for changes in DNA UKp68 damage response signaling using immunofluorescence (IF) and western blotting for H2AX, a marker of DNA damage signaling and DSB23. Treatment of HeLa cells with 500?nM JQ1 for 12?h led to increased nuclear H2AX immunostaining (Fig.?1a). This increase in DNA damage signaling corresponded to increased DSB formation as quantified using the neutral comet single cell gel electrophoresis assay (Fig.?1b), increased cleavage of PARP (cPARP), an indicator of apoptosis (Fig.?1c), and subsequent growth inhibition (Fig.?1d). The increase in DNA damage signaling, DSB formation, apoptosis, and growth inhibition following treatment with JQ1 was also seen in HCT116 cells (Supplementary Fig.?1aCd). Open in a separate window Fig. 1 BRD4 bromodomain inhibition causes DNA damage and apoptosis.a Left panel: Immuno-fluorescence (IF) images of H2AX fluorescence in HeLa cells following treatment with DMSO or 500?nM JQ1 for 12?h (test (**test (****test (*test (**test (****test (***transcription (Fig.?2b)19. Despite potent suppression of MYC, treatment with triptolide alone did not result in increased DNA damage signaling, DSBs, or apoptosis in cells, while treatment with ARV-825 alone was again associated with increased DNA damage, PARP-mediated apoptosis and DSB formation over the same time course (Fig.?2a, c, d). These findings suggest a mechanism of DNA damage and apoptosis induction following BRD4 loss that is independent of changes in MYC transcription Deramciclane alone, which has been reported as a predominant mechanism responsible for the decreased survival of oncogenic cells following treatment with BET bromodomain inhibitors20. Cells pretreated with triptolide followed by co-treatment with ARV-825 showed abrogation of DNA damage signaling, DSB formation, and apoptosis (Fig.?2a, c, d, respectively), suggesting that DNA Deramciclane damage caused by BRD4 loss requires the presence of active transcription bubbles. Deramciclane Abrogation of BET bromodomain degrader-induced DNA damage and DSB formation by triptolide was also seen in HCT116 cells (Supplementary Fig.?2a, b, respectively). It should be noted that the short course of RNAPII inhibition.
