Cell-Penetrating CEBPB and CEBPD Leucine Zipper Decoys as Broadly Acting Anti-Cancer Agents.

Transcription factors are key players underlying cancer formation, growth, survival, metastasis and treatment resistance, yet few drugs exist to directly target them. Here, we characterized the in vitro and in vivo anti-cancer efficacy of novel synthetic cell-penetrating peptides (Bpep and Dpep) designed to interfere with the formation of active leucine-zipper-based dimers by CEBPB and CEBPD, transcription factors implicated in multiple malignancies. Both peptides similarly promoted apoptosis of multiple tumor lines of varying origins, without such effects on non-transformed cells. Combined with other treatments (radiation, Taxol, chloroquine, doxorubicin), the peptides acted additively to synergistically and were fully active on Taxol-resistant cells. The peptides suppressed expression of known direct CEBPB/CEBPD targets IL6, IL8 and asparagine synthetase (ASNS), supporting their inhibition of transcriptional activation. Mechanisms by which the peptides trigger apoptosis included depletion of pro-survival survivin and a required elevation of pro-apoptotic BMF. Bpep and Dpep significantly slowed tumor growth in mouse models without evident side effects. Dpep significantly prolonged survival in xenograft models. These findings indicate the efficacy and potential of Bpep and Dpep as novel agents to treat a variety of cancers as mono- or combination therapies.

Sex differences in gut microbiota modulation of aversive conditioning, open field activity, and basolateral amygdala dendritic spine density.

Gut microbiota influence numerous aspects of host biology, including brain structure and function. Growing evidence implicates gut microbiota in aversive conditioning and anxiety-related behaviors, but research has focused almost exclusively on males. To investigate whether effects of gut dysbiosis on aversive learning and memory differ by sex, adult female and male C57BL/6N mice were orally administered a moderate dose of nonabsorbable antimicrobial medications (ATMs: neomycin, bacitracin, and pimaricin) or a control over 10 days. Changes in gut microbiome composition were analyzed by 16S rRNA sequencing. Open field behavior, cued aversive learning, context recall, and cued recall were assessed. Following behavioral testing, the morphology of basolateral amygdala (BLA) principal neuron dendrites and spines was characterized. Results revealed that ATMs induced gut dysbiosis in both sexes, with stronger effects in females. ATMs also exerted sex-specific effects on behavior and neuroanatomy. Males were more susceptible than females to microbial modulation of locomotor activity and anxiety-like behavior. Females were more susceptible than males to ATM-induced impairments in aversive learning and cued recall. Context recall remained intact, as did dendritic structure of BLA principal neurons. However, ATMs exerted a sex-specific effect on spine density. A second experiment was conducted to isolate the effects of gut perturbation to cued recall. Extinction was also examined. Results revealed no effect of ATMs on cued recall or extinction, suggesting that gut dysbiosis preferentially impacts aversive learning. These data shed new light on how gut microbiota interact with sex to influence aversive conditioning, open field behavior, and BLA dendritic spine architecture.

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.