Role of BET family of transcription regulators in DNA replication stress

BET proteins function as epigenetic readers, and BET inhibition has been shown to have activity against many different cancers. BET inhibitors have been fast-tracked into first clinical trials, however the effects of these inhibitors are still poorly understood. A more detailed understanding about the physiological effects of BET inhibition is important and may lead to improved clinical applications as well as allowing safer use of BET inhibitor drugs. In this thesis we have been able to identify that BET inhibition unexpectedly leads to an increase in RNA synthesis that is associated with conflicts between transcription and replication, leading to replication fork slowing, a sign of replication stress. We have identified BRD4 as the main target of BET inhibition in this process which is needed for normal fork progression. Interestingly, our data suggest that increased RNA synthesis requires free P-TEFb to be released from its inhibitor complex HEXIM1 to allow increased RNA polymerase II phosphorylation. HEXIM1 is required for BET inhibitor-induced replication-transcription conflicts. We have shown that BET inhibitor-induced fork slowing does not activate the canonical ATR-Chk1 replication stress response pathway. However, it activates the homologous recombination factor RAD51, which is recruited into nuclear foci in response to BET inhibitor treatment. RAD51 depletion followed by BET inhibition prevents replication fork slowing but activates the replication stress response. HEXIM1 depletion has the same effect, preventing fork slowing and activating the replication stress response. These data suggest that i) replication fork slowing is required to prevent DNA damage formation in presence of BET inhibitors and ii) this depends on HEXIM1, which is upstream of transcription-replication conflicts, and RAD51, which acts downstream of transcription-replication conflicts. Our data shed light on the initial stress response during the first 8 hours of BET inhibition. They implicate HEXIM1 and RAD51, which both play potential roles in BET inhibitor resistance, in the BET inhibitor-induced replication stress pathway

BET inhibition induces HEXIM1- and RAD51-dependent conflicts between transcription and replication

Summary: BET bromodomain proteins are required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on processes such as DNA replication will be important for future clinical applications. Here, we show that BET inhibition, and specifically inhibition of BRD4, causes replication stress through a rapid overall increase in RNA synthesis. We provide evidence that BET inhibition acts by releasing P-TEFb from its inhibitor HEXIM1, promoting interference between transcription and replication. Unusually, these transcription-replication conflicts do not activate the ATM/ATR-dependent DNA damage response but recruit the homologous recombination factor RAD51. Both HEXIM1 and RAD51 promote BET inhibitor-induced fork slowing but also prevent a DNA damage response. Our data suggest that BET inhibitors slow replication through concerted action of transcription and recombination machineries and shed light on the importance of replication stress in the action of this class of experimental cancer drugs. : Bowry et al. show that BET inhibitors, emerging cancer therapeutics that target transcription programs, cause conflicts between transcription and replication by deregulating P-TEFb. These conflicts recruit the homologous recombination factor RAD51, which slows down replication and suppresses DNA damage. This highlights the importance of replication stress for BET inhibitor treatment. Keywords: JQ1, I-BET151, BRD4, P-TEFb, homologous recombination, replication fork, replication stress, DNA damage, cance