ARTICLE ABSTRACTInhibitors of the bromodomain and extraterminal domain (BET) protein family attenuate the proliferation of several tumor cell lines. These effects are mediated, at least in part, through repression of c-MYC. In colorectal cancer, overexpression of c-MYC due to hyperactive WNT/β-catenin/TCF signaling is a key driver of tumor progression; however, effective strategies to target this oncogene remain elusive. Here, we investigated the effect of BET inhibitors (BETi) on colorectal cancer cell proliferation and c-MYC expression. Treatment of 20 colorectal cancer cell lines with the BETi JQ1 identified a subset of highly sensitive lines. JQ1 sensitivity was higher in cell lines with microsatellite instability but was not associated with the CpG island methylator phenotype, c-MYC expression or amplification status, BET protein expression, or mutation status of TP53, KRAS/BRAF, or PIK3CA/PTEN. Conversely, JQ1 sensitivity correlated significantly with the magnitude of c-MYC mRNA and protein repression. JQ1-mediated c-MYC repression was not due to generalized attenuation of β-catenin/TCF-mediated transcription, as JQ1 had minimal effects on other β-catenin/TCF target genes or β-catenin/TCF reporter activity. BETi preferentially target super-enhancer–regulated genes, and a super-enhancer in c-MYC was recently identified in HCT116 cells to which BRD4 and effector transcription factors of the WNT/β−catenin/TCF and MEK/ERK pathways are recruited. Combined targeting of c-MYC with JQ1 and inhibitors of these pathways additively repressed c-MYC and proliferation of HCT116 cells. These findings demonstrate that BETi downregulate c-MYC expression and inhibit colorectal cancer cell proliferation and identify strategies for enhancing the effects of BETi on c-MYC repression by combinatorial targeting the c-MYC super-enhancer. Mol Cancer Ther; 15(6); 1217–26. ©2016 AACR.