Dominant-Negative ATF5 Compromises Cancer Cell Survival by Targeting CEBPB and CEBPD

The basic leucine zipper transcription factor ATF5 is overexpressed in many tumor types and interference with its expression or function inhibits cancer cell survival. As a potential therapeutic approach to exploit these findings, we created dominant-negative (DN) ATF5 forms lacking DNA-binding ability that retain the ATF5 leucine zipper, and thus associate with and sequester ATF5's requisite leucine zipper–binding partners. Preclinical studies with DN-ATF5, including a cell-penetrating form, show in vitro and in vivo efficacy in compromising cancer cell survival. However, DN-ATF5's targets, and particularly those required for tumor cell survival, have been unknown. We report that cells lacking ATF5 succumb to DN-ATF5, indicating that ATF5 itself is not DN-ATF5's obligate target. Unbiased pull-down assays coupled with mass spectrometry and immunoblotting revealed that DN-ATF5 associates in cells with the basic leucine zipper proteins CEBPB and CEBPD and coiled-coil protein CCDC6. Consistent with DN-ATF5 affecting tumor cell survival by suppressing CEBPB and CEBPD function, DN-ATF5 interferes with CEBPB and CEBPD transcriptional activity, while CEBPB or CEBPD knockdown promotes apoptotic death of multiple cancer cells lines, but not of normal astrocytes. We propose a two-pronged mechanism by which DN-ATF5 kills tumor cells. One is by inhibiting heterodimer formation between ATF5 and CEBPB and CDBPD, thus suppressing ATF5-dependent transcription. The other is by blocking the formation of transcriptionally active CEBPB and CEBPD homodimers as well as heterodimers with partners in addition to ATF5. Implications: This study indicates that the potential cancer therapeutic DN-ATF5 acts by associating with and blocking the transcriptional activities of CEBPB and CEBPD.