Abstract Myc family of transcription factors are deregulated in most cancers and their expression projects a poor prognosis. Genetic and pharmacological inhibition of Myc and several Myc targets leads to tumor regression and apoptosis. Recently, inhibitors of BET Bromodomain proteins (BETi) were shown to have anti-tumor properties and this has been attributed to Myc down-regulation. Here we show that two structurally distinct BETi affects replication and cell cycle progression at low concentrations where the transcriptome remains largely unaltered. Thymidine incorporation and flow cytometry analyses demonstrates that S-phase progression is hindered at these concentrations. However, in a cell-free system replication is not impaired suggesting that BETi-mediated block of replication is linked to effects on chromatin. Furthermore, at higher concentration of BETi, S-phase entry of cells is completely abrogated. Ectopic expression of Myc fails to rescue these phenotypes, suggesting a novel function of BET bromodomain proteins in replication and cell cycle regulation. Our pharmacogenetic screen in the presence of sub-lethal doses of BETi has identified several small molecule inhibitors to synergize with BETi to enhance apoptosis of Myc driven tumors. Citation Format: Somsundar Veppil Muralidharan, Joydeep Bhadury, Lydia Green, Lisa M. Nilsson, Kevin G. McLure, Jonas A. Nilsson. Effects of BET bromodomain inhibitors on replication and cell cycle progression. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B44.